iGEM 2017 — Team Kyoto

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Kyoto/Notebook

* [![](https://static.igem.org/mediawiki/2017/4/4a/Kyototitle222.png)](https://2017.igem.org/Team%3AKyoto)
* ![](https://static.igem.org/mediawiki/2017/6/6e/松５.png)
* [Project](https://2017.igem.org/team%3AKyoto/Project)
  + [Description](https://2017.igem.org/team%3AKyoto/Project)
  + [Results](https://2017.igem.org/Team%3AKyoto/Results)
  + [Demonstrate](https://2017.igem.org/Team%3AKyoto/Demonstrate)
  + [Discussion](https://2017.igem.org/Team%3AKyoto/Discussion)
  + [Basic Parts](https://2017.igem.org/Team%3AKyoto/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3AKyoto/Composite_Part)
* [Practices](https://2017.igem.org/Team%3AKyoto/Human_Practices)
  + [Human Practices](https://2017.igem.org/Team%3AKyoto/Human_Practices)
  + [Integrated Practices](https://2017.igem.org/Team%3AKyoto/Integrated_Practices)
  + [Public Engagement](https://2017.igem.org/Team%3AKyoto/Engagement)
  + [Collaborations](https://2017.igem.org/Team%3AKyoto/Collaborations)
  + [Events](https://2017.igem.org/Team%3AKyoto/Events)
  + [Further Consideration](https://2017.igem.org/Team%3AKyoto/Discussion_HP)
* [Experiment](https://2017.igem.org/Team%3AKyoto/Notebook)
  + [Notebook](https://2017.igem.org/Team%3AKyoto/Notebook)
  + [Material&Methods](https://2017.igem.org/Team%3AKyoto/Experiments)
* [Team](https://2017.igem.org/Team%3AKyoto/Team)
  + [Members](https://2017.igem.org/Team%3AKyoto/Team)
  + [Attributions](https://2017.igem.org/Team%3AKyoto/Attributions)
* [Criteria](https://2017.igem.org/Team%3AKyoto/Achievements)
  + [Achievements](https://2017.igem.org/Team%3AKyoto/Achievements)
  + [HP/Silver](https://2017.igem.org/Team%3AKyoto/HP/Silver)
  + [HP/Gold](https://2017.igem.org/Team%3AKyoto/HP/Gold_Integrated)

[![](https://static.igem.org/mediawiki/2017/c/c5/Kyoto_notebook_jump.png)](#kyoto-calender)

# Notebook

| July | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | 1 | 2 | 3 | 4 | 5 |
| 6 | 7 | 8 | 9 | [10](#0710) | [11](#0711) | [12](#0712) |
| [13](#0713) | [14](#0714) | [15](#0715) | [16](#0716) | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 |  |  |

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | 1 | 2 | 3 | 4 | 5 |
| [6](#0806) | [7](#0807) | [8](#0808) | [9](#0809) | [10](#0810) | [11](#0811) | [12](#0812) |
| [13](#0813) | [14](#0814) | [15](#0815) | 16 | [17](#0817) | [18](#0818) | [19](#0819) |
| [20](#0820) | [21](#0821) | [22](#0822) | [23](#0823) | [24](#0824) | 25 | [26](#0826) |
| [27](#0827) | [28](#0828) | [29](#0829) | 30 | [31](#0831) |  |  |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | [1](#0901) | [2](#0902) | [3](#0903) | [4](#0904) | [5](#0905) |
| [6](#0906) | [7](#0907) | [8](#0908) | [9](#0909) | [10](#0910) | 11 | 12 |
| 13 | [14](#0914) | [15](#0915) | [16](#0916) | [17](#0917) | [18](#0918) | [19](#0919) |
| [20](#0920) | [21](#0921) | [22](#0922) | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 |  |  |

| October | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | 1 | 2 | 3 | [4](#1004) | [5](#1005) |
| [6](#1006) | [7](#1007) | 8 | [9](#1009) | 10 | 11 | [12](#1012) |
| [13](#1013) | [14](#1014) | [15](#1015) | 16 | [17](#1017) | [18](#1018) | [19](#1019) |
| [20](#1020) | 21 | [22](#1022) | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 |  |  |

### 07/10

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | shortAK2 | shortAK2 | B.xylophilus cDNA |
| 2 | Top1 | Top1 | B.xylophilus cDNA |
| 3 | long AK2 | long AK2 | B.xylophilus cDNA |
| 4 | eef-1g | eef-1g | B.xylophilus cDNA |
| 5 | AK1 | AK1 | B.xylophilus cDNA |
| 6 | Asb | Asb | B.xylophilus cDNA |
| 7 | 14-3-3zeta | 14-3-3zeta | B.xylophilus cDNA |
| 8 | tropomiosin | tropomiosin | B.xylophilus cDNA |
| 9 | 14-3-3 protein | 14-3-3 protein | B.xylophilus cDNA |

PCR Polymerase : 5\*Q5 Polymerase
98.0°C : 30sec
98.0°C : 10sec
58.0°C : 30sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle
PCR pulification: lane 2~9
Gel extraction: lane 2~9

### 07/11

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | 2 | no data | no data | 237.8 |
| 2 | 3 | no data | no data | 180.5 |
| 3 | 4 | no data | no data | 185.6 |
| 4 | 5 | no data | no data | 167.2 |
| 5 | 6 | no data | no data | 328.2 |
| 6 | 7 | no data | no data | 133.7 |
| 7 | 8 | no data | no data | 168.4 |
| 8 | 9 | no data | no data | 242.5 |

The way of extraction : Gel extraction

### 07/11

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 1 | Template2 | 1.0 |
| 2 | YEplac195/sma1 | 1 | Template3 | 1.0 |
| 3 | YEplac195/sma1 | 1 | Template4 | 1.0 |
| 4 | YEplac195/sma1 | 1 | Template5 | 1.0 |
| 5 | YEplac195/sma1 | 1 | Template6 | 1.0 |
| 6 | YEplac195/sma1 | 1 | Template7 | 1.0 |
| 7 | YEplac195/sma1 | 1 | Template8 | 1.0 |
| 8 | YEplac195/sma1 | 1 | Template9 | 1.0 |

### 07/11

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | 7/11 ligation product |

### 07/12

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | pRS315 | 1 | Template | 1.0 |

### 07/12

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | 7/12 ligation product 2 |
| 2 | DH5a | 7/12 ligation product 3 |
| 3 | DH5a | 7/12 ligation product 4 |
| 4 | DH5a | 7/12 ligation product 5 |
| 5 | DH5a | 7/12 ligation product 6 |
| 6 | DH5a | 7/12 ligation product 7 |
| 7 | DH5a | 7/12 ligation product 8 |
| 8 | DH5a | 7/12 ligation product 9 |

### 07/13

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | T7Top1 | T7Top1 | 7/10 gel extraction product2 |
| 2 | T7long AK2 | T7long AK2 | 7/10 gel extraction product3 |
| 3 | T7eef-1g | T7eef-1g | 7/10 gel extraction product4 |
| 4 | T7AK1 | T7AK1 | 7/10 gel extraction product5 |
| 5 | T7Asb | T7Asb | 7/10 gel extraction product6 |
| 6 | T714-3-3zeta | T714-3-3zeta | 7/10 gel extraction product7 |
| 7 | T7tropomiosin | T7tropomiosin | 7/10 gel extraction product8 |
| 8 | T714-3-3 protein | T714-3-3 protein | 7/10 gel extraction product9 |

PCR Polymerase : ExTaq
98.0°C : 1min
98.0°C : 10sec
58.0°C : 30sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

### 07/14

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | 1 | 1.84 | 1.96 | 98.4 |
| 2 | 2 | 1.79 | 1.85 | 76.2 |
| 3 | 3 | 1.85 | 2.1 | 102.1 |
| 4 | 4 | 1.62 | 0.49 | 9.5 |
| 5 | 5 | 1.91 | 1.91 | 83.6 |
| 6 | 6 | 1.82 | 1.73 | 48.0 |
| 7 | 7 | 1.85 | 1.85 | 83.0 |
| 8 | 8 | 1.86 | 2.84 | 81.5 |
| 9 | 9 | 1.57 | 0.33 | 22.6 |
| 10 | 10 | 1.54 | 0.55 | 17.8 |
| 11 | 11 | 1.83 | 2.02 | 88.0 |
| 12 | 12.0 | 1.85 | 2.94 | 82.0 |
| 13 | 13.0 | 1.83 | 1.83 | 90.6 |
| 14 | 14.0 | 1.87 | 1.65 | 46.2 |
| 15 | 15.0 | 1.89 | 2.0 | 75.8 |
| 16 | 16.0 | 1.67 | 1.98 | 114.0 |
| 17 | 17.0 | 1.88 | 1.46 | 88.5 |
| 18 | 18.0 | 1.85 | 1.91 | 99.4 |
| 19 | 19.0 | 1.79 | 1.79 | 53.8 |
| 20 | 20.0 | 1.37 | 0.58 | 17.0 |
| 21 | 21.0 | 1.63 | 0.57 | 0.57 |
| 22 | 22.0 | 1.83 | 1.84 | 53.0 |

The way of extraction : promega

### 07/15

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 1 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 2 | 2 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 3 | 3 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 4 | 4 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 5 | 5 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 6 | 6 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 7 | 7 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 8 | 8 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 9 | 9 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 10 | 10 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 11 | 11 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 12 | 12 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 13 | 13 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 14 | 14 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 15 | 15 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 16 | 16 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 17 | 17 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 18 | 18 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 19 | 19 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 20 | 20 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 21 | 21 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |
| 22 | 22 | 5.0 | Pst1 | 0.05 | Sac1 | 0.05 | 10xM | 1.0 | 10.0 |

### 07/16

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | T7 3 | 1.86 | 2.28 | 255.7 |
| 2 | T7 4 | 1.83 | 2.13 | 121.9 |
| 3 | T7 5 | 1.83 | 2.29 | 224.2 |
| 4 | T7 6 | 1.85 | 2.18 | 146.1 |
| 5 | T7 7 | 1.85 | 2.25 | 96.5 |
| 6 | T7 8 | 1.85 | 2.27 | 165.4 |
| 7 | T7 9 | No data | No data | No data |

The way of extraction : promega

### 07/16

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | short AK2 | short AK2 | B.xylophilus cDNA |
| 2 | Top1 | Top1 | B.xylophilus cDNA |

PCR Polymerase : Extaq
94.0°C : 1min
94.0°C : 30sec
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/06

#### Nebuilder

Yoshimoto

| Sample | Vectorname | Vector amount (ul) | Insert1 name | Insert1 amount(ul) | Insert2 name | Insert2 amount (ul) | Mastermix amount (ul) | Total amount(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 2.5 | Gal-AK | 0.8 | Ter-AK | 1.0 | 10.0 | 20.0 |
| 2 | YEplac195/sma1 | 2.5 | GPD-AK | 1.2 | Ter-AK | 1.0 | 10.0 | 20.0 |

### 08/06

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | Insert |

### 08/07

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | 1 | 1.97 | 2.03 | 36.0 |
| 2 | 2 | 2.03 | 1.89 | 28.7 |
| 3 | 3 | 1.92 | 1.98 | 34.3 |
| 4 | 4 | 1.96 | 1.88 | 35.5 |
| 5 | 5 | 2.03 | 1.87 | 16.2 |
| 6 | 6 | 2.01 | 2.11 | 50.0 |
| 7 | 7 | 1.95 | 1.95 | 30.3 |
| 8 | 8 | 1.95 | 2.06 | 38.2 |
| 9 | 9 | 2.12 | 1.94 | 17.0 |
| 10 | 10 | no data | no data | no data |
| 11 | 11 | 1.99 | 1.92 | 27.9 |
| 12 | 12 | 1.96 | 1.79 | 19.1 |
| 13 | 13 | 1.95 | 1.96 | 29.4 |
| 14 | 14 | 1.98 | 1.64 | 22.7 |

The way of extraction : fastgene plasmid mini kit

### 08/08

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | 8/6 Nebuilder product |
| 2 | DH5a | 8/6 Nebuilder product |
| 3 | DH5a | 8/6 Nebuilder product |
| 4 | DH5a | 8/6 Nebuilder product |
| 5 | DH5a | 8/6 Nebuilder product |
| 6 | DH5a | 8/6 Nebuilder product |
| 7 | DH5a | 8/6 Nebuilder product |
| 8 | DH5a | 8/6 Nebuilder product |

### 08/08

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | miniprep 1 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 2 | miniprep 2 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 3 | miniprep 3 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 4 | miniprep 4 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 5 | miniprep 5 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 6 | miniprep 7 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |

### 08/09

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | 8 | 1.69 | 1.32 | 12.1 |
| 2 | 9 | 1.97 | 1.45 | 17.0 |
| 3 | 11 | 1.83 | 0.99 | 12.5 |
| 4 | 12 | 1.92 | 1.05 | 12.3 |

The way of extraction : promega PureYield Plasmid Midiprepkit

### 08/09

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 8/8 miniprep8 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 2 | 8/8 miniprep9 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 3 | 8/8 miniprep11 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |
| 4 | 8/8 miniprep12 | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/c/c0/IGKU_b1_027_0.jpeg)

### 08/09

#### Restriction Enzyme digestion

Fukuda

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | AK2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 2 | AK2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 3 | eef-1g | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 4 | eef-1g | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 5 | AK1 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 6 | AK1 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 7 | Asb | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 8 | Asb | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 9 | 14-3-3-zeta | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 10 | 14-3-3-zeta | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 11 | tropomiosin | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 12 | tropomiosin | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 13 | 14-3-3 protein | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |
| 14 | 14-3-3 protein | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 6.0 | 36.0 |

### 08/10

#### Nebuilder

Yoshimoto

| Sample | Vectorname | Vector amount (ul) | Insert1 name | Insert1 amount(ul) | Insert2 name | Insert2 amount (ul) | Mastermix amount (ul) | Total amount(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 2.5 | Gal-AK | 0.8 | Ter-AK | 1.0 | 10.0 | 20.0 |
| 2 | YEplac195/sma1 | 2.5 | GPD-AK | 1.2 | Ter-AK | 1.0 | 10.0 | 20.0 |

### 08/10

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | insert |

### 08/10

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | T7AK2F | T7AK2R | B.xylophils cDNA1 |
| 2 | T7AK2F | T7AK2R | B.xylophils cDNA2 |
| 3 | T7Egf-1gF | T7Egf-1gR | B.xylophils cDNA3 |
| 4 | T7Egf-1gF | T7Egf-1gR | B.xylophils cDNA4 |
| 5 | T7AK1F | T7Ak1R | B.xylophils cDNA5 |
| 6 | T7AK1F | T7Ak1R | B.xylophils cDNA6 |
| 7 | T714-3-3zetaF | T714-3-3 zetaR | B.xylophils cDNA7 |
| 8 | T714-3-3zetaF | T714-3-3 zetaR | B.xylophils cDNA8 |
| 9 | T7tropomiosin F | T7tropomiosin R | B.xylophils cDNA9 |
| 10 | T7tropomiosin F | T7tropomiosin R | B.xylophils cDNA10 |
| 11 | T714-3-3 F | T714-3-3 R | B.xylophils cDNA11 |
| 12 | T714-3-3 F | T714-3-3 R | B.xylophils cDNA12 |
| 13 | T7AK2F | T7AK2R | water |

PCR Polymerase : gotaq
94.0°C : 2min
94.0°C : 30sec
62.0°C : 30sec
68.0°C : 1min20sec
68.0°C : 7min
4.0°C : Infinate
X30 cycle

![](https://static.igem.org/mediawiki/2017/2/21/IGKU_b2_029_0.jpeg)

### 08/11

#### colony\_PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 1 |
| 2 | M13F | M13R | 3 |
| 3 | M13F | M13R | 5 |
| 4 | M13F | M13R | 7 |
| 5 | M13F | M13R | 9 |
| 6 | M13F | M13R | 11 |
| 7 | M13F | M13R | 13 |
| 8 | M13F | M13R | 15 |
| 9 | M13F | M13R | 17 |
| 10 | M13F | M13R | 19 |
| 11 | M13F | M13R | 21 |
| 12 | M13F | M13R | 23 |
| 13 | M13F | M13R | 25 |
| 14 | M13F | M13R | 27 |
| 15 | M13F | M13R | 29 |
| 16 | M13F | M13R | 31 |
| 17 | M13F | M13R | 33 |
| 18 | M13F | M13R | 35 |
| 19 | M13F | M13R | 37 |
| 20 | M13F | M13R | 39 |
| 21 | M13F | M13R | 41 |
| 22 | M13F | M13R | 43 |
| 23 | M13F | M13R | 45 |
| 24 | M13F | M13R | 47 |
| 25 | M13F | M13R | 49 |

![](http://https://2017.igem.org/File:IGKU_b1_030_0.jpeg)

PCR Polymerase : QuickTaq
94.0°C : 2min
94.0°C : 30sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

### 08/11

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 1-1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 2 | 1-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 3 | 1-3 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 4 | 1-4 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 5 | 1-5 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 6 | 2-1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 7 | 1-6 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 8 | 2-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 9 | 2-3 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 10 | 2-4 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 11 | 2-5 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 12 | 2-6 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 13 | 3-1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 14 | 3-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 15 | 3-3 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |
| 16 | 3-4 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 3.0 |

![](https://static.igem.org/mediawiki/2017/a/af/IGKU_b2_030_0.jpeg)

### 08/11

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | 1 | 1.88 | 1.6 | 116.8 |
| 2 | 8 | 1.9 | 1.67 | 170.0 |
| 3 | 9 | 1.91 | 1.71 | 167.6 |
| 4 | 12 | 1.88 | 1.6 | 135.6 |

The way of extraction : promega PureYield Plasmid Midiprepkit

### 08/11

#### colony\_PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 2 |
| 2 | M13F | M13R | 4 |
| 3 | M13F | M13R | 6 |
| 4 | M13F | M13R | 8 |
| 5 | M13F | M13R | 10 |
| 6 | M13F | M13R | 12 |
| 7 | M13F | M13R | 14 |
| 8 | M13F | M13R | 16 |
| 9 | M13F | M13R | 18 |
| 10 | M13F | M13R | 20 |
| 11 | M13F | M13R | 22 |
| 12 | M13F | M13R | 24 |
| 13 | M13F | M13R | 26 |
| 14 | M13F | M13R | 28 |
| 15 | M13F | M13R | 30 |
| 16 | M13F | M13R | 32 |
| 17 | M13F | M13R | 34 |
| 18 | M13F | M13R | 36 |
| 19 | M13F | M13R | 38 |
| 20 | M13F | M13R | 40 |
| 21 | M13F | M13R | 42 |
| 22 | M13F | M13R | 44 |
| 23 | M13F | M13R | 46 |
| 24 | M13F | M13R | 48 |
| 25 | M13F | M13R | 50 |
| 26 | M13F | M13R | 53 |
| 27 | M13F | M13R | 54 |
| 28 | M13F | M13R | 55 |
| 29 | M13F | M13R | 56 |

PCR Polymerase : Quick\_taq
94.0°C : 2min
94.0°C : 30sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 10min
4.0°C : Infinate
X35 cycle

### 08/11

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 1 | AK2-gal1 | 1.0 |
| 2 | YEplac195/sma1 | 1 | AK2-GPD | 1.0 |

### 08/11

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 7A | 5.0 | Pst1 | 0.5 | 10xH | 6.0 | 60.0 |
| 2 | 7B | 5.0 | Pst1 | 0.5 | 10xH | 6.0 | 60.0 |

![](https://static.igem.org/mediawiki/2017/f/f1/IGKU_b2_032_0.jpeg)

### 08/11

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | AK2-gal1 Nebuilder |
| 2 | M13F | M13R | AK2 GPD Nebuilder |
| 3 | M13F | M13R | gfp gal Nebuilder |
| 4 | M13F | M13R | gfp GPD Nebuilder |
| 5 | M13F | M13R | AK1 gal Nebuilder |
| 6 | M13F | M13R | AK1 GPD Nebuilder |

PCR Polymerase : 2\*KAPA 2G Robust HS Readymix
94.0°C : 2min
94.0°C : 15sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 1min
4.0°C : Infinate
X35 cycle

### 08/11

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | insert |

### 08/12

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | AK1F | AK1R | 8 |
| 2 | AK1F | AK1R | 9 |
| 3 | AK1F | AK1R | 12 |
| 4 | AK1F | AK1R | 8/6 gal |
| 5 | AK1F | AK1R | 8/6 GPD |
| 6 | AK1F | AK1R | gal1 AK1 |
| 7 | AK1F | AK1R | gal1 AK1 |
| 8 | AK1F | AK1R | water |

PCR Polymerase : quicktaq
94.0°C : 2min
94.0°C : 30sec
60.0°C : 30sec
68.0°C : 1min
68.0°C : 7min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/8/85/IGKU_b2_033_0.jpeg)

### 08/12

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 1 | gblock 71 | 1.0 |
| 2 | YEplac195/sma1 | 1 | gblock 72 | 1.0 |
| 3 | YEplac195/sma1 | 1 | gblock 73 | 1.0 |
| 4 | YEplac195/sma1 | 1 | gblock 74 | 1.0 |
| 5 | YEplac195/sma1 | 1 | gblock 75 | 1.0 |
| 6 | YEplac195/sma1 | 1 | gblock 76 | 1.0 |
| 7 | YEplac195/sma1 | 1 | gblock 77 | 1.0 |
| 8 | YEplac195/sma1 | 1 | gblock 78 | 1.0 |

### 08/12

#### Transformation

Yoshimito

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | insert |

### 08/13

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | AK2F | AK2F | 8 |
| 2 | AK2F | AK2F | 9 |
| 3 | AK2F | AK2F | 12 |
| 4 | AK2F | AK2F | 8/6 gal |
| 5 | AK2F | AK2F | 8/6 GPD |
| 6 | AK2F | AK2F | 7 |
| 7 | AK2F | AK2F | water |

PCR Polymerase : 2\*KAPA hifi HS ready mix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 2min
72.0°C : 2min
4.0°C : Infinate
X40 cycle

![](https://static.igem.org/mediawiki/2017/0/08/IGKU_b2_034_0.jpeg)

### 08/13

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 2 | 2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 3 | 3 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 4 | 4 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 5 | 5 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 6 | 7 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 7 | 1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 8 | 7 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 9 | 9 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 10 | 8 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 11 | 12 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/3/31/IGKU_b2_035_0.jpeg)

### 08/13

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 14 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 2 | 16 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 3 | 22 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 4 | 24 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 5 | 26 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 6 | 30 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 7 | 36 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 8 | 24 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 9 | 14 | 5.0 | Pst1 | 0.0 | 10xH | 0.0 | 10.0 |
| 10 | 22 | 5.0 | Pst1 | 0.0 | 10xH | 0.0 | 10.0 |
| 11 | 36 | 5.0 | Pst1 | 0.0 | 10xH | 0.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/a/ad/IGKU_b2_036_0.jpeg)

### 08/13

#### colony PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 77-1 |
| 2 | M13F | M13R | 77-2 |
| 3 | M13F | M13R | 77-3 |
| 4 | M13F | M13R | 78-4 |
| 5 | M13F | M13R | 78-5 |

PCR Polymerase : 2\*KAPA hifi HS ready mix
95.0°C : 1min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

### 08/13

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | 14 | 1.91 | 1.44 | 82.6 |
| 2 | 16 | 1.96 | 1.67 | 91.4 |
| 3 | 22 | 2.04 | 1.77 | 96.7 |
| 4 | 24 | 1.88 | 1.5 | 93.0 |
| 5 | 26 | 1.89 | 1.51 | 89.4 |
| 6 | 30 | 1.9 | 1.48 | 74.6 |
| 7 | 36 | 1.91 | 1.5 | 87.5 |

The way of extraction : promega PureYield Plasmid Miniprepkit

### 08/13

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 1 | Gal-AK2 99 | 2.0 |
| 2 | YEplac195/sma1 | 1 | GPD-AK2 80 | 2.0 |
| 3 | YEplac195/sma1 | 1 | Ter-AK2 81 | 2.0 |
| 4 | YEplac195/sma1 | 1 | T7 AK1 82 | 2.0 |
| 5 | YEplac195/sma1 | 1 | T7 AK1.2 83 | 2.0 |
| 6 | YEplac195/sma1 | 1 | Gal1 gfp 85 | 2.0 |
| 7 | YEplac195/sma1 | 1 | ter gfp 86 | 2.0 |

### 08/13

#### Nebuilder

Yoshimoto

| Sample | Vectorname | Vector amount (ul) | Insert1 name | Insert1 amount(ul) | Insert2 name | Insert2 amount (ul) | Mastermix amount (ul) | Total amount(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | YEPlac195/sma1 | 0.25 | GPD-AK | 1.5 | Ter-AK | 5.0 | 5.0 | 12.0 |
| 2 | YEPlac195/sma1 | 0.25 | Gal-AK | 2.0 | Ter-AK | 5.0 | 5.0 | 12.0 |

### 08/13

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | Nebuilder product |
| 2 | DH5a | Nebuilder product |
| 3 | DH5a | Nebuilder product |
| 4 | DH5a | Nebuilder product |

### 08/14

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 79-1 |
| 2 | M13F | M13R | 80-1 |
| 3 | M13F | M13R | 80-2 |
| 4 | M13F | M13R | 81-1 |
| 5 | M13F | M13R | 97-1 |
| 6 | M13F | M13R | 98-1 |
| 7 | M13F | M13R | 98-2 |
| 8 | M13F | M13R | 98-3 |
| 9 | M13F | M13R | 92-1 |
| 10 | M13F | M13R | 99-1 |
| 11 | M13F | M13R | 100-1 |
| 12 | M13F | M13R | 101-1 |
| 13 | M13F | M13R | 101-2 |
| 14 | M13F | M13R | 101-3 |
| 15 | M13F | M13R | 102-1 |
| 16 | M13F | M13R | water |

PCR Polymerase : 2\*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 1min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/1/1f/IGKU_b2_038_0.jpeg)![](https://static.igem.org/mediawiki/2017/f/fe/IGKU_b2_038_1.jpeg)

### 08/14

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 2 | 3 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 3 | 4 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 4 | 5 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 5 | 7 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 6 | 14 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 7 | 16 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 8 | 22 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 9 | 77-1 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 10 | 77-2 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 11 | 77-3 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 12 | 24 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 13 | 26 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 14 | 30 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 15 | 36 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 16 | 78-1 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 17 | 78-2 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 18 | 78 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 19 | 99 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 20 | 100 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 21 | 101 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/b/bf/IGKU_b2_039_0.jpeg)![](https://static.igem.org/mediawiki/2017/c/cf/IGKU_b2_039_1.jpeg)

### 08/14

#### colony\_PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 97 |
| 2 | M13F | M13R | 98 |
| 3 | M13F | M13R | 99 |
| 4 | M13F | M13R | 100 |
| 5 | M13F | M13R | 101 |
| 6 | M13F | M13R | 102 |

PCR Polymerase : 2\*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/14

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | Nebuilder product gal1-gfp |
| 2 | DH5a | Nebuilder product GPD-gfp |
| 3 | DH5a | 8/13 ligation product Gal-AK2 |
| 4 | DH5a | 8/13 ligation product GPD-AK2 |
| 5 | DH5a | 8/13 ligation product Ter-AK2 |

### 08/14

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | ligation product Gal-AK2 |
| 2 | DH5a | ligation product GPD-AK2 |
| 3 | DH5a | ligation product Ter-AK2 |

### 08/14

#### Miniprep\_Nanodrop

Yoshimoto

| sample | Sample name | 260/280 | 260/230 | ng/ul |
| --- | --- | --- | --- | --- |
| 1 | 101 | 1.97 | 0.93 | 24.9 |
| 2 | 100 | 1.99 | 0.99 | 34.8 |
| 3 | 97 | 1.94 | 1.35 | 50.1 |
| 4 | 98 | 1.97 | 1.97 | 40.3 |
| 5 | 99.0 | 1.96 | 1.65 | 99.7 |
| 6 | 73.5 | 1.82 | 0.9 | 47.9 |
| 7 | 78.4 | 1.86 | 0.99 | 43.7 |
| 8 | 71-3 | 1.88 | 1.21 | 67.4 |
| 9 | 36 | 1.88 | 1.41 | 129.5 |
| 10 | 77-1 | 1.81 | 0.99 | 70.8 |
| 11 | 77-2 | 1.89 | 1.2 | 63.0 |
| 12 | 30 | 1.82 | 1.23 | 135.8 |
| 13 | 26 | 1.86 | 1.31 | 143.9 |
| 14 | 24 | 1.88 | 1.43 | 141.5 |
| 15 | 14 | 1.82 | 1.31 | 178.7 |
| 16 | 16 | 1.91 | 1.37 | 132.3 |
| 17 | 22 | 1.79 | 1.35 | 207.8 |
| 18 | 7 | 1.85 | 1.28 | 94.4 |
| 19 | 5 | 1.79 | 0.91 | 63.8 |
| 20 | 4 | 1.66 | 0.81 | 64.2 |
| 21 | 2 | 1.87 | 0.96 | 50.5 |
| 22 | 3 | 1.79 | 1.15 | 67.1 |

The way of extraction : promega

### 08/15

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 92 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 2 | 97 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 3 | 98 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 4 | 100 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 5 | 101 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 6 | 92 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 7 | 97 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 8 | 98 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 9 | 100 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 10 | 101 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/2/26/IGKU_b2_040_0.jpeg)

### 08/15

#### Nebuilder

Yoshimoto

| Sample | Vectorname | Vector amount (ul) | Insert1 name | Insert1 amount(ul) | Insert2 name | Insert2 amount (ul) | Mastermix amount (ul) | Total amount(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 0.25 | Gal-AK | 1.825 | Ter-AK | 1.5 | 5.0 | 10.0 |
| 2 | YEplac195/sma1 | 0.25 | GPD-AK | 1.25 | Ter-AK | 1.5 | 5.0 | 10.0 |

### 08/15

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | DH5a | 111 |
| 2 | DH5a | 112 |

### 08/17

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK25 | IK26 | Rev |
| 2 | IK27 | IK22 | yeast genome |
| 3 | IK23 | IK28 | yeast genome |

PCR Polymerase : 2\*KAPA hifi HS Ready mix
98.0°C : 3min
98.0°C : 20sec
62.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/c/ce/IGKU_b2_041_0.jpeg)

### 08/17

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK27 | IK22 | yeast genome |
| 2 | IK23 | IK28 | yeast genome |
| 3 | IK27 | IK28 | GPDp+Rev+GPDt |

PCR Polymerase : 2\*KAPA hifi HS ready mix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/9/96/IGKU_b2_042_0.jpeg)

### 08/17

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK25 | IK26 | Rev |
| 2 | IK27 | IK22 | yeast genome |
| 3 | IK23 | IK28 | yeast genome |
| 4 | IK25 | IK26 | Rev after purification |
| 5 | IK27 | IK22 | GPDp after purification |
| 6 | IK23 | IK28 | GPDt after purification |

PCR Polymerase : 2\*KAPA hifi HS ready mix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X36 cycle

![](https://static.igem.org/mediawiki/2017/b/bf/IGKU_b2_043_0.jpeg)

### 08/17

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 86-1 |
| 2 | M13F | M13R | 86-2 |
| 3 | M13F | M13R | 81-1 |
| 4 | M13F | M13R | 81-2 |
| 5 | M13F | M13R | 81-3 |
| 6 | M13F | M13R | 95-1 |
| 7 | M13F | M13R | 95-2 |
| 8 | M13F | M13R | 95-3 |
| 9 | M13F | M13R | 95-4 |
| 10 | M13F | M13R | 93-1 |
| 11 | M13F | M13R | 93-2 |
| 12 | M13F | M13R | 92 |
| 13 | M13F | M13R | brue colony |
| 14 | M13F | M13R | 11 |

![](http://https://static.igem.org/mediawiki/2017/4/48/IGKU_b1_045_0.jpeg)

PCR Polymerase : 2\*KAPA hifi HS readymix
95.0°C : 3min
95.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 1min
4.0°C : Infinate
X35 cycle

### 08/17

#### Nebuilder

Yoshimoto

| Sample | Vectorname | Vector amount (ul) | Insert1 name | Insert1 amount(ul) | Insert2 name | Insert2 amount (ul) | Mastermix amount (ul) | Total amount(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 0.8 | Gal-AK2 | 6.5 | Ter-AK2 | 5.0 | 10.0 | 22.3 |
| 2 | YEplac195/sma1 | 0.8 | GPD-AK2 | 4.2 | Ter-AK2 | 5.0 | 10.0 | 20.0 |

### 08/17

#### Transformation

Yoshimoto

| sample | CompetentCell name | DNA name |
| --- | --- | --- |
| 1 | BL21 | 113 |
| 2 | BL21 | 114 |
| 3 | BL21 | 115 |

### 08/18

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 79-2 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 2 | 79-3 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 3 | 79-6 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 4 | 80-2 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 5 | 80-7 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 6 | 80-13 | 5.0 | Pst1 | 1.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 7 | 81-1 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 8 | 81-2 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 9 | 97 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 10 | 99-A | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 11 | 99-B | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 12 | 100 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 13 | 100-1 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 14 | 100-2 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 15 | 101-1 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 16 | 101 3 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |
| 17 | 102 | 5.0 | Pst1 | 1.0 | None | None | 10xH | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/1/12/IGKU_b2_044_0.jpeg)![](https://static.igem.org/mediawiki/2017/e/e1/IGKU_b2_044_1.jpeg)![](https://static.igem.org/mediawiki/2017/b/b3/IGKU_b2_044_2.jpeg)

### 08/18

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK27 | IK28 | Rev |

PCR Polymerase : 2\*KAPA hifi HS Ready mix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 20sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/1/16/IGKU_b2_045_0.jpeg)

### 08/18

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | AK2R | 79-2 |
| 2 | M13F | AK2R | 79-3 |
| 3 | M13F | AK2R | 80-13 |
| 4 | M13F | AK2F | 79-2 |
| 5 | M13F | AK2F | 79-3 |
| 6 | M13F | AK2F | 80-13 |
| 7 | M13R | M13F | blue colony |

PCR Polymerase : 2\*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/18

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK22 | IK27 | GPDp after purification |
| 2 | IK22 | IK27 | GPDp after purification |
| 3 | IK22 | IK27 | GPDp after purification |
| 4 | IK22 | IK27 | GPDp after purification |
| 5 | M13F | AK2R | 113gal |
| 6 | M13F | AK2R | 114GPD |

PCR Polymerase : 2\*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane1~4
Gel extraction: lane1~4

![](https://static.igem.org/mediawiki/2017/4/4c/IGKU_b2_047_0.jpeg)

### 08/19

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | 141 | 0.34 | 2T7akRRE3' | 9.4 |
| 2 | 142 | 0.34 | 2T7ak1stem | 8.3 |
| 3 | 143 | 0.34 | 1T7ak1stem | 4.4 |
| 4 | 144 | 0.34 | 2T7RREak1 | 5.3 |
| 5 | 145 | 0.34 | 1T7RREak1 | 8.5 |
| 6 | 146 | 0.34 | GPD+AK2 | 13.2 |
| 7 | 147 | 0.34 | Gal1+AK2 | 15.8 |
| 8 | 148 | 0.34 | AK2+ter | 20.1 |

### 08/20

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK22 | IK27 | GPDp |
| 2 | IK22 | IK27 | GPDp |
| 3 | IK22 | IK27 | yeast genome |
| 4 | IK22 | IK27 | yeast genome |
| 5 | IK22 | IK27 | yeast genome |
| 6 | IK22 | IK27 | yeast genome |
| 7 | IK27 | IK28 | GPDp+Rev+GPDt |
| 8 | IK27 | IK28 | GPDp+Rev+GPDt |

PCR Polymerase : 2\*KAPA hifi HS ready mix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/20

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK27 | IK22 | GPDp |
| 2 | IK21 | IK22 | GPDp |

PCR Polymerase : 2\*KAPA HS readymix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/8/88/IGKU_b2_052_0.jpeg)

### 08/21

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK29 | IK30 | Rev |
| 2 | IK29 | IK30 | Rev |
| 3 | IK29 | IK30 | Rev |
| 4 | IK29 | IK30 | Rev |
| 5 | M13R | AK1R | 141 1-1 |
| 6 | M13R | AK1R | 141 1-2 |
| 7 | M13R | AK1R | 141 1-3 |
| 8 | M13R | AK1R | 141 1-4 |
| 9 | M13R | AK1R | 142 1-1 |
| 10 | M13R | AK1R | 142 1-2 |
| 11 | M13R | AK1R | 142 1-3 |
| 12 | M13R | AK1R | 142 1-4 |
| 13 | M13R | AK1F | 141 1-1 |
| 14 | M13R | AK1F | 141 1-2 |
| 15 | M13R | AK1F | 141 1-3 |
| 16 | M13R | AK1F | 141 1-4 |
| 17 | M13R | AK1F | 142 1-1 |
| 18 | M13R | AK1F | 142 1-2 |
| 19 | M13R | AK1F | 142 1-3 |
| 20 | M13R | AK1F | 142 1-4 |

PCR Polymerase : 2\*KAPA hifi HS Readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/0/00/IGKU_b2_055_0.jpeg)

### 08/21

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | AK1R | 141 2-1 |
| 2 | M13F | AK1R | 141 2-4 |
| 3 | M13F | AK1R | 141 1-15 |
| 4 | M13F | AK1R | 142 1-5 |
| 5 | M13F | AK1R | 143 9 |
| 6 | M13F | AK1R | 143 13 |
| 7 | M13F | AK1R | 143 27 |
| 8 | M13F | AK1R | 143 2 |
| 9 | M13F | AK1F | 141 2-1 |
| 10 | M13F | AK1F | 141 2-4 |
| 11 | M13F | AK1F | 141 1-15 |
| 12 | M13F | AK1F | 142 1-5 |
| 13 | M13F | AK1F | 143 9 |
| 14 | M13F | AK1F | 143 13 |
| 15 | M13F | AK1F | 143 27 |
| 16 | M13F | AK1F | 143 2 |

PCR Polymerase : 2\*KAPA hifi HS Readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/c/cf/IGKU_b2_056_0.jpeg)

### 08/21

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK29 | IK30 | Rev |
| 2 | IK29 | IK30 | Rev |
| 3 | IK29 | IK30 | Rev |
| 4 | IK29 | IK30 | Rev |

PCR Polymerase : 2\*KAPA hifi HS Readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane2~4
Gel extraction: lane2~4

![](https://static.igem.org/mediawiki/2017/d/de/IGKU_b2_057_0.jpeg)

### 08/22

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | GPDp |
| 2 | IK21 | IK22 | GPDp |
| 3 | IK21 | IK22 | GPDp |
| 4 | IK21 | IK22 | GPDp |
| 5 | IK21 | IK22 | water |
| 6 | IK23 | IK28 | GPDt |
| 7 | IK23 | IK28 | GPDt |
| 8 | IK23 | IK28 | GPDt |
| 9 | IK23 | IK28 | GPDt |
| 10 | IK23 | IK28 | water |

PCR Polymerase : 2\*KAPA hifi HS Readymix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane2~4
Gel extraction: lane2~4

![](https://static.igem.org/mediawiki/2017/9/9a/IGKU_b2_058_0.jpeg)

### 08/22

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK27 | IK28 | GPDp+Rev+GPDt |
| 2 | IK27 | IK28 | GPDp+Rev+GPDt |
| 3 | IK23 | IK24 | yeast genome |
| 4 | IK23 | IK24 | yeast genome |
| 5 | IK23 | IK24 | yeast genome |
| 6 | IK23 | IK24 | yeast genome |
| 7 | IK21 | IK24 | GPDp+Rev+GPDt |

PCR Polymerase : 2\*KAPA hifi HS ReadyMIx
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/1/1a/IGKU_b2_059_0.jpeg)

### 08/22

#### PCR

Fukuda

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 144 11 |
| 2 | M13F | M13R | 144 3 |
| 3 | M13F | M13R | 144 7 |
| 4 | M13F | M13R | 144 1 |
| 5 | M13F | M13R | 144 17 |
| 6 | M13F | M13R | 144 5 |
| 7 | M13F | M13R | 144 9 |
| 8 | M13F | M13R | 144 13 |
| 9 | M13F | M13R | 142 1 |
| 10 | M13F | M13R | 142 5 |
| 11 | M13F | M13R | 142 3 |
| 12 | M13F | M13R | 142 4 |
| 13 | M13F | M13R | 142 2-1 |
| 14 | M13F | M13R | 142 2-2 |
| 15 | M13F | M13R | 142 2-3 |
| 16 | M13F | M13R | 142 2-4 |
| 17 | M13F | M13R | 143 13 |
| 18 | M13F | M13R | 143 15 |
| 19 | M13F | M13R | 143 5 |
| 20 | M13F | M13R | 153 21 |
| 21 | M13F | M13R | 143 17 |
| 22 | M13F | M13R | 143 19 |
| 23 | M13F | M13R | 143 25 |
| 24 | M13F | M13R | 143 9 |

PCR Polymerase : 2\*KAPA hifi HS ReadyMix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/22

#### PCR

Takimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 145 1-13 |
| 2 | M13F | M13R | 145 2-13 |
| 3 | M13F | M13R | 145 2-15 |
| 4 | M13F | M13R | 145 1-5 |
| 5 | M13F | M13R | 145 2-17 |
| 6 | M13F | M13R | 145 2-11 |
| 7 | M13F | M13R | 145 2-9 |
| 8 | M13F | M13R | 145 1-17 |
| 9 | M13F | M13R | 146 2-3 |
| 10 | M13F | M13R | 146 2-1 |
| 11 | M13F | M13R | 146 1-3 |
| 12 | M13F | M13R | 146 2-7 |
| 13 | M13F | M13R | 146 2-9 |
| 14 | M13F | M13R | 146 2-5 |
| 15 | M13F | M13R | 146 1-4 |

PCR Polymerase : 2\*KAPA hifi HS ReadyMix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/22

#### Nebuilder

Yoshimoto

| Sample | Vectorname | Vector amount (ul) | Insert1 name | Insert1 amount(ul) | Insert2 name | Insert2 amount (ul) | Mastermix amount (ul) | Total amount(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 171.0 | Vector amount (ul) | Gal1+gfp-2 | 0.78 | Gal1+gfp | 1.03 | 10.0 | Total amount(ul) |
| 2 | 172.0 | Vector amount (ul) | Gal1+AK2 | 1.26 | Gal1+AK2-2 | 1.01 | 10.0 | Total amount(ul) |
| 3 | 173.0 | Vector amount (ul) | 164.0 | 0.65 | 165.0 | 0.57 | 10.0 | Total amount(ul) |
| 4 | 174.0 | Vector amount (ul) | 161.0 | 0.65 | 162.0 | 0.76 | 10.0 | Total amount(ul) |
| 5 | 175.0 | Vector amount (ul) | 161.0 | 0.38 | 167.0 | 0.83 | 10.0 | Total amount(ul) |
| 6 | 176.0 | Vector amount (ul) | 166.0 | 0.38 | 165.0 | 0.83 | 10.0 | Total amount(ul) |
| 7 | 177.0 | Vector amount (ul) | 163.0 | 0.38 | 162.0 | 0.83 | 10.0 | Total amount(ul) |
| 8 | 178.0 | Vector amount (ul) | 163.0 | 0.38 | 167.0 | 0.83 | 10.0 | Total amount(ul) |

### 08/22

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | None | None | GPDp+Rev+GPDt |
| 2 | IK27 | IK28 | lane1 after 15 cycle |

PCR Polymerase : 2\*KAPA hifi Hs ReadyMix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/22

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK23 | IK28 | GPDt |
| 2 | IK23 | IK28 | GPDt |
| 3 | IK23 | IK28 | GPDt |
| 4 | IK23 | IK28 | GPDt |
| 5 | IK27 | IK30 | GPDp + Rev |
| 6 | IK21 | IK30 | GPDp + Rev |

PCR Polymerase : 2\*KAPA hifi Robust
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/d/d2/IGKU_b2_069_0.jpeg)

### 08/23

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 147 2-17 |
| 2 | M13F | M13R | 147 1-2 |
| 3 | M13F | M13R | 147 2-3 |
| 4 | M13F | M13R | 147 7 |
| 5 | M13F | M13R | 141 1-1 |
| 6 | M13F | M13R | 141 1-2 |
| 7 | M13F | M13R | 141 1-4 |
| 8 | M13F | M13R | 148 2-3 |
| 9 | M13F | M13R | 148 2-5 |
| 10 | M13F | M13R | 148 2-4 |
| 11 | M13F | M13R | 148 1-5 |
| 12 | M13F | M13R | 148 1-3 |
| 13 | M13F | M13R | 144 11 |
| 14 | M13F | M13R | 144 3 |
| 15 | M13F | M13R | 144 1 |
| 16 | M13F | M13R | 146 1-6 |
| 17 | M13F | M13R | 146 1-4 |
| 18 | M13F | M13R | 146 2-5 |
| 19 | M13F | M13R | 146 2-7 |
| 20 | M13F | M13R | 142 2-2 |
| 21 | M13F | M13R | 142 1-1 |
| 22 | M13F | M13R | 142 2-7 |
| 23 | M13F | M13R | 142 2-3 |
| 24 | M13F | M13R | 145 2-11 |
| 25 | M13F | M13R | 145 1-17 |
| 26 | M13F | M13R | 145 2-9 |
| 27 | M13F | M13R | 145 2-17 |
| 28 | M13F | M13R | 143 5 |
| 29 | M13F | M13R | 143 17 |
| 30 | M13F | M13R | 143 15 |
| 31 | M13F | M13R | 143 21 |
| 32 | M13F | AK1R | 147 2-17 |
| 33 | M13F | AK1R | 147 1-2 |
| 34 | M13F | AK1R | 147 2-3 |
| 35 | M13F | AK1R | 147 7 |
| 36 | M13F | AK1R | 141 1-1 |
| 37 | M13F | AK1R | 141 1-2 |
| 38 | M13F | AK1R | 141 1-4 |
| 39 | M13F | AK1R | 148 2-3 |
| 40 | M13F | AK1R | 148 2-5 |
| 41 | M13F | AK1R | 148 2-4 |
| 42 | M13F | AK1R | 148 1-5 |
| 43 | M13F | AK1R | 148 1-3 |
| 44 | M13F | AK1R | 144 11 |
| 45 | M13F | AK1R | 144 3 |
| 46 | M13F | AK1R | 144 1 |
| 47 | M13F | AK1R | 146 1-6 |
| 48 | M13F | AK1R | 146 1-4 |
| 49 | M13F | AK1R | 146 2-5 |
| 50 | M13F | AK1R | 146 2-7 |
| 51 | M13F | AK1R | 142 2-2 |
| 52 | M13F | AK1R | 142 1-1 |
| 53 | M13F | AK1R | 142 2-7 |
| 54 | M13F | AK1R | 142 2-3 |
| 55 | M13F | AK1R | 145 2-11 |
| 56 | M13F | AK1R | 145 1-17 |
| 57 | M13F | AK1R | 145 2-9 |
| 58 | M13F | AK1R | 145 2-17 |
| 59 | M13F | AK1R | 143 5 |
| 60 | M13F | AK1R | 143 17 |
| 61 | M13F | AK1R | 143 15 |
| 62 | M13F | AK1R | 143 21 |

PCR Polymerase : 2\*KAPA hifi robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/e/e9/IGKU_b2_068_0.jpeg)![](https://static.igem.org/mediawiki/2017/7/7d/IGKU_b2_068_1.jpeg)![](https://static.igem.org/mediawiki/2017/5/50/IGKU_b2_068_2.jpeg)![](https://static.igem.org/mediawiki/2017/f/f2/IGKU_b2_068_3.jpeg)

### 08/24

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 141 2-1 |
| 2 | M13F | M13R | 141 2-2 |
| 3 | M13F | M13R | 141 2-4 |
| 4 | M13F | M13R | 141 2-6 |
| 5 | M13F | M13R | 142 1-5 |
| 6 | M13F | M13R | 142 2-4 |
| 7 | M13F | M13R | 142 1-3 |
| 8 | M13F | M13R | 142 1-4 |
| 9 | M13F | M13R | 143 13 |
| 10 | M13F | M13R | 143 19 |
| 11 | M13F | M13R | 143 25 |
| 12 | M13F | M13R | 144 18 |
| 13 | M13F | M13R | 144 17 |
| 14 | M13F | M13R | 144 5 |
| 15 | M13F | M13R | 144 9 |
| 16 | M13F | M13R | 143 19 |
| 17 | M13F | M13R | 145 1-5 |
| 18 | M13F | M13R | 145 2-15 |
| 19 | M13F | M13R | 143 2-13 |
| 20 | M13F | M13R | 145 1-13 |
| 21 | M13F | AK1R | 141 2-1 |
| 22 | M13F | AK1R | 141 2-2 |
| 23 | M13F | AK1R | 141 2-4 |
| 24 | M13F | AK1R | 141 2-6 |
| 25 | M13F | AK1R | 142 1-5 |
| 26 | M13F | AK1R | 142 2-4 |
| 27 | M13F | AK1R | 142 1-3 |
| 28 | M13F | AK1R | 142 1-4 |
| 29 | M13F | AK1R | 145 1-5 |
| 30 | M13F | AK1R | 145 2-15 |
| 31 | M13F | AK1R | 143 2-13 |
| 32 | M13F | AK1R | 145 1-13 |
| 33 | M13F | AK1R | 144 17 |
| 34 | M13F | AK1R | 144 5 |
| 35 | M13F | AK1R | 144 9 |
| 36 | M13F | AK1R | 143 19 |
| 37 | M13F | AK1R | 143 13 |
| 38 | M13F | AK1R | 143 19 |
| 39 | M13F | AK1R | 143 25 |
| 40 | M13F | AK1R | 144 9 |

PCR Polymerase : 2\*KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/4/4c/IGKU_b2_070_0.jpeg)

### 08/26

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK27 | IK30 | GPDp + Rev after gel extraction |
| 2 | IK27 | IK30 | GPDp + Rev after gel extraction |
| 3 | IK27 | IK30 | GPDp + Rev after gel extraction |
| 4 | IK27 | IK30 | GPDp + Rev after gel extraction |
| 5 | IK23 | IK28 | GPDt after PCR purification |
| 6 | IK23 | IK28 | GPDt after PCR purification |
| 7 | IK23 | IK28 | GPDt after PCR purification |
| 8 | IK23 | IK28 | GPDt after PCR purification |

PCR Polymerase : 2\*KAPA hifi HS Readymix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/4/40/IGKU_b2_072_0.jpeg)

### 08/26

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 145-2 11 |
| 2 | M13F | M13R | 145-1 9 |
| 3 | M13F | M13R | 145 2-7 |
| 4 | M13F | M13R | 145 1-21 |
| 5 | M13F | M13R | 145 1-3 |
| 6 | M13F | M13R | 145 2-3 |
| 7 | M13F | M13R | 145 1-19 |
| 8 | M13F | M13R | 142 1-2 |
| 9 | M13F | AK1F | 145-2 11 |
| 10 | M13F | AK1F | 145-1 9 |
| 11 | M13F | AK1F | 145 2-7 |
| 12 | M13F | AK1F | 145 1-21 |
| 13 | M13F | AK1F | 145 1-3 |
| 14 | M13F | AK1F | 145 2-3 |
| 15 | M13F | AK1F | 145 1-19 |
| 16 | M13F | AK1F | 142 1-2 |

PCR Polymerase : 2\*KAPA hifi
98.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 25sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

### 08/26

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 114-1 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 2 | 114-2 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 3 | 172 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 4 | 172-1 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 5 | 172-2 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 6 | 172-4 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 7 | 175-3 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 8 | 175-10 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 9 | 176 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 10 | 177 | 2.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |

### 08/27

#### Nebuilder

Yoshimoto

| Sample | Vectorname | Vector amount (ul) | Insert1 name | Insert1 amount(ul) | Insert2 name | Insert2 amount (ul) | Mastermix amount (ul) | Total amount(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Rev | 3.02 | GPDp+Rev | 0.669 | GPDt | 0.875 | 10.0 | Total amount(ul) |
| 2 | 173.0 | 3.02 | 164.0 | 0.325 | 165.0 | 0.285 | 5.0 | Total amount(ul) |
| 3 | 175.0 | 3.02 | 161.0 | 0.19 | 167.0 | 0.42 | 5.0 | Total amount(ul) |
| 4 | 178.0 | 3.02 | 163.0 | 0.41 | 167.0 | 0.42 | 5.0 | Total amount(ul) |

### 08/27

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 171-1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 2 | 171-1-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 3 | 172-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 4 | 172-2-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 5 | 172-3 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 6 | 172-4 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 7 | 172-5 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 8 | 172-6 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 9 | 172-8 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 10 | 172-9 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 11 | 172-10 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 12 | 172-11 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 13 | 172-12 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 14 | 172-13 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 15 | 175-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 16 | 175-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 17 | 175-3 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 18 | 175-4 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 19 | 176-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 20 | 177-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 21 | 177-3 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 22 | 177-4 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 23 | 177-5 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 24 | 177-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 25 | 177-1-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 26 | 171-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 27 | 172-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 28 | 175-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 29 | 175-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 30 | 175-3 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 31 | 175-4 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 32 | 176-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 33 | 176-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 34 | 177-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 35 | 177-3 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 36 | 177-4 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 37 | 177-5 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |

### 08/27

#### Restriction Enzyme digestion

Ohara

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 171 1-1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 2 | 171 1-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 3 | 171 2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 4 | 171 3 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 5 | 172-1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 6 | 172-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 7 | 172-3 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 8 | 172-4-1 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 9 | 172-4-2 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 10 | 172-5 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 11 | 172-6 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 12 | 172-8 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 13 | 172-9 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 14 | 172-10 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 15 | 172-11 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 16 | 172-12 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 17 | 172-13 | 5.0 | Pst1 | 1.0 | 10xH | 1.0 | 10.0 |
| 18 | 171-1-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 19 | 174-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 20 | 175-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 21 | 175-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 22 | 175-3 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 23 | 175-4 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 24 | 176-1 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 25 | 176-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 26 | 177-2 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 27 | 177-3 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 28 | 177-4 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |
| 29 | 177-5 | 5.0 | EcoR1 | 1.0 | 10xH | 1.0 | 10.0 |

### 08/28

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 145-2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 2 | 142 2-11 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 3 | 142 2-2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 4 | 142 2-1 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 5 | 142 2-3 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 6 | 242 1-4 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 7 | 142 2-4 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 8 | 142 1-5 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 9 | 142 1-3 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 10 | 142 2-5 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 11 | 142 1-2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 12 | 142 1-1 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 13 | 145 2-3 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 14 | 145 1-2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 15 | 145 2-7 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 16 | 145 4-9 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 17 | 145 1-11 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 18 | 145 1-3 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 19 | 145 2-11 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 20 | 145 2-5 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 21 | 145 2-8 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 22 | 145 1-14 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 23 | 145 2-15 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 24 | 145 2- 4-2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 25 | 145 1-17 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 26 | 145 2-17 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |

### 08/29

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 142 1-3 | 5.0 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul 1ul | 10.0 |
| 2 | 145 1-11 | 5.0 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul 1ul | 9.0 |
| 3 | 145 1-3 | 5.0 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul 1ul | 9.0 |
| 4 | 145 2-42 | 5.0 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul 1ul | 9.0 |

### 08/29

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK27 | IK28 | Rev, GPDp, GPDt |
| 2 | IK27 | IK28 | Rev+GPDp, GPDt |
| 3 | IK27 | IK28 | Rev plasmid |
| 4 | IK27 | IK28 | Rev plasmid |
| 5 | IK27 | IK28 | Rev plasmid |
| 6 | IK27 | IK28 | Rev plasmid |

PCR Polymerase : KAKA Robust
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 08/31

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 141 1-2 | 3.5 | EcoR1 | 0.5 | Xba1 | 0.5 | 10xN | 1.0 | 10.0 |
| 2 | 141 1-2 | 3.5 | EcoR1 | 0.5 | Xba1 | 0.5 | 10xN | 1.0 | 10.0 |
| 3 | 141 1-3 | 3.5 | EcoR1 | 0.5 | Xba1 | 0.5 | 10xN | 1.0 | 10.0 |
| 4 | 141 1-3 | 3.5 | EcoR1 | 0.5 | Xba1 | 0.5 | 10xN | 1.0 | 10.0 |
| 5 | 144-9 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 6 | 144-9 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 7 | 144 17 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 8 | 144 17 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 9 | 144 3 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 10 | 144 3 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 11 | 144 3 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 12 | 144 1 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 13 | 144 1 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 14 | 144 1 | 3.5 | EcoR1 | 0.5 | Not1 | 0.5 | 10xH, BSA | 1ul, 1ul | 10.0 |
| 15 | 143 21 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 16 | 143 21 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 17 | 143 21 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 18 | 143 15 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 19 | 143 15 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 20 | 143 15 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 21 | 143 15 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 22 | 143 5 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 23 | 143 5 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 24 | 143 5 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 25 | 143 13 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 26 | 145 1-3 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 27 | 145 1-3 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 28 | 145 1-3 | 3.5 | Xba1 | 0.5 | Not1 | 0.5 | 10xK, BSA | 1ul, 1ul | 10.0 |
| 29 | 142 2-2 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 30 | 142 1-3 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 31 | 142 | 5.0 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 32 | 142 2-2 | 5.0 | Not1 | 0.5 | Xba1 | 0.5 | 10xK BSA | 1ul, 1ul | 10.0 |
| 33 | 142 1-3 | 5.0 | Not1 | 0.5 | Xba1 | 0.5 | 10xK BSA | 1ul, 1ul | 10.0 |
| 34 | 142 | 5.0 | Not1 | 0.5 | Xba1 | 0.5 | 10xK BSA | 1ul, 1ul | 10.0 |

### 09/01

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | P1 |
| 2 | M13F | M13R | P2 |
| 3 | M13F | M13R | P3 |
| 4 | M13F | M13R | P4 |
| 5 | M13F | M13R | T1 |
| 6 | M13F | M13R | T2 |
| 7 | M13F | M13R | T3 |
| 8 | M13F | M13R | T4 |

PCR Polymerase : KAPA
98.0°C : 3min
98.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 10min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/f/ff/IGKU_b2_083_0.jpeg)

### 09/02

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 144-3 | 3.5 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 2 | 142 | 3.5 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 3 | 145 1-11 | 3.5 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/d/df/IGKU_b2_085_0.jpeg)![](https://static.igem.org/mediawiki/2017/8/84/IGKU_b2_085_1.jpeg)

### 09/02

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 165-1 |
| 2 | M13F | M13R | 165-2 |
| 3 | M13F | M13R | 165-3 |
| 4 | M13F | M13R | 165-4 |
| 5 | M13F | IK60 | 165-1 |
| 6 | M13F | IK60 | 165-2 |
| 7 | M13F | IK60 | 165-3 |
| 8 | M13F | IK60 | 165-4 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/1/18/IGKU_b2_086_0.jpeg)

### 09/02

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 142 2-2 | 3.5 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 2 | 1421 | 3.5 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |
| 3 | 142 1-3 | 3.5 | Xba1 | 0.5 | EcoR1 | 0.5 | 10xM | 1.0 | 10.0 |

### 09/03

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 0.35 | 161 | 6.19 |
| 2 | YEplac195/sma1 | 0.35 | 162 | 12.2 |
| 3 | YEplac195/sma1 | 0.35 | 163 | 13.1 |
| 4 | YEplac195/sma1 | 0.35 | 164 | 10.32 |
| 5 | YEplac195/sma1 | 0.35 | 166 | 17.25 |
| 6 | YEplac195/sma1 | 0.35 | 167 | 13.28 |

### 09/03

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | IK55 | GFPgalp-1 |
| 2 | M13F | IK55 | GFPgalp-2 |
| 3 | M13F | IK55 | GFPgalp-3 |
| 4 | M13F | IK55 | GFPgalp-4 |
| 5 | M13F | IK55 | GFPt-1 |
| 6 | M13F | IK55 | GFPt-2 |
| 7 | M13F | IK55 | GFPt-3 |
| 8 | M13F | IK55 | GFPt-4 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 09/04

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | GPDp |
| 2 | IK21 | IK22 | GPDp |
| 3 | IK21 | IK22 | GPDp |
| 4 | IK21 | IK22 | GPDp |
| 5 | IK23 | IK28 | GPDter |
| 6 | IK23 | IK28 | GPDter |
| 7 | IK23 | IK28 | GPDter |
| 8 | IK23 | IK28 | GPDter |
| 9 | IK29 | IK30 | Rev long |
| 10 | IK29 | IK30 | Rev long |
| 11 | IK29 | IK30 | Rev long |
| 12 | IK29 | IK30 | Rev long |
| 13 | IK21 | IK28 | GPDter(PCRp), Rev(PCRp), GPDp(gel ex) |
| 14 | IK21 | IK28 | GPDter(PCRp), Rev+GPDp(PCRp) |

PCR Polymerase : KAPA
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~4
PCR pulification: 9~12
Gel extraction: 1~4
Gel extraction: 9~12

![](https://static.igem.org/mediawiki/2017/8/8e/IGKU_b2_090_0.jpeg)

### 09/04

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | AK1F | 142-2-1 |
| 2 | IK60 | AK1F | 142-2-2 |
| 3 | IK60 | AK1F | 142-2-3 |
| 4 | IK60 | AK1F | 142-2-4 |
| 5 | IK60 | AK1F | 142-2-5 |
| 6 | IK60 | AK1F | 142-1-1 |
| 7 | IK60 | AK1F | 142-1-2 |
| 8 | IK60 | AK1F | 142-1-3 |
| 9 | IK60 | AK1F | 142-1-4 |
| 10 | IK60 | AK1F | 142-1-5 |
| 11 | IK60 | M13F | 144-3 |
| 12 | IK60 | M13R | 145-1-11 |
| 13 | IK60 | AK1F | 144-3 |
| 14 | IK60 | AK1F | 145-1-11 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/3/32/IGKU_b2_091_0.jpeg)

### 09/04

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | M13R | 142-2-1 |
| 2 | IK60 | M13R | 142-2-2 |
| 3 | IK60 | M13R | 142-2-3 |
| 4 | IK60 | M13R | 142-2-4 |
| 5 | IK60 | M13R | 142-2-5 |
| 6 | IK60 | M13R | 142-1-1 |
| 7 | IK60 | M13R | 142-1-2 |
| 8 | IK60 | M13R | 142-1-3 |
| 9 | IK60 | M13R | 142-1-4 |
| 10 | IK60 | M13R | 142-1-5 |
| 11 | M13F | M13R | gfpp |
| 12 | IK55 | M13R | gfpp |
| 13 | M13F | M13R | gfpt M13 |
| 14 | IK55 | M13R | gfp 55 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 20sec
61.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/5/5b/IGKU_b2_092_0.jpeg)

### 09/04

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK27 | IK30 | Rev+p |
| 2 | IK21 | IK30 | Rev+p |
| 3 | IK27 | IK22 | GPDp |
| 4 | IK27 | IK22 | GPDp |
| 5 | IK27 | IK22 | GPDp |
| 6 | IK21 | IK22 | GPDp |
| 7 | IK21 | IK22 | GPDp |
| 8 | IK21 | IK22 | GPDp |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

### 09/04

#### PCR

Fukuda

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | eef-1g F | eef-1gR | 3 |
| 2 | eef-1g F | eef-1gR | 4 |
| 3 | AK1F | AK1R | 5 |
| 4 | AK1F | AK1R | 6 |
| 5 | asbF | asbR | 7 |
| 6 | asbF | asbR | 8 |

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/6/64/IGKU_b2_106_0.jpeg)![](https://static.igem.org/mediawiki/2017/8/8b/IGKU_b2_106_1.jpeg)

### 09/05

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 161 |
| 2 | M13F | M13R | 161 |
| 3 | M13F | M13R | 162 |
| 4 | M13F | M13R | 163 |
| 5 | M13F | M13R | 166 |
| 6 | M13F | M13R | 167 |
| 7 | M13F | M13R | 168 |
| 8 | M13F | M13R | 142 |
| 9 | IK60 | M13R | 161 |
| 10 | IK60 | M13R | 161 |
| 11 | M13F | IK60 | 162 |
| 12 | IK60 | M13R | 163 |
| 13 | IK60 | M13R | 166 |
| 14 | M13F | IK60 | 167 |
| 15 | M13F | IK60 | 168 |
| 16 | M13F | IK60 | 142 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 20sec
61.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/9/95/IGKU_b2_093_0.jpeg)

### 09/05

#### Restriction Enzyme digestion

Yoshimto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | gfpp | 5.0 | EcoRI | 2.0 | None | None | H | 1.0 | 10.0 |
| 2 | gfpp | 5.0 | Not1 | 1.0 | HindⅢ | 1.0 | K | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/c/c8/IGKU_b2_094_0.jpeg)

### 09/05

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK28 | Rev, p, t |
| 2 | IK21 | IK28 | Rev+p, t |
| 3 | IK27 | IK28 | Rev, p, t |
| 4 | IK27 | IK28 | Rev+p, t |
| 5 | IK21 | IK24 | Rev, p, t |
| 6 | IK21 | IK24 | Rev+p, t |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/b/b2/IGKU_b2_095_0.jpeg)

### 09/05

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK30 | 9/5 PCR p Rev,pro |
| 2 | IK21 | IK30 | 9/5 PCR p Rev,pro |
| 3 | IK21 | IK30 | 9/5 PCR p Rev,pro |
| 4 | IK21 | IK30 | 9/5 PCR p Rev,pro |
| 5 | IK29 | IK24 | 9/5 PCR p Rev,ter |
| 6 | IK29 | IK24 | 9/5 PCR p Rev,ter |
| 7 | IK29 | IK24 | 9/5 PCR p Rev,ter |
| 8 | IK29 | IK24 | 9/5 PCR p Rev,ter |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle
PCR pulification: 5.0
Gel extraction: 5.0

![](https://static.igem.org/mediawiki/2017/0/03/IGKU_b2_097_0.jpeg)![](https://static.igem.org/mediawiki/2017/b/b4/IGKU_b2_097_1.jpeg)

### 09/05

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | GPD1promoter |
| 2 | IK21 | IK22 | GPD1promoter |
| 3 | IK21 | IK22 | GPD1promoter |
| 4 | IK21 | IK22 | GPD1promoter |
| 5 | IK21 | IK22 | GPD1promoter |

PCR Polymerase : 2\*KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/1/11/IGKU_b2_100_0.jpeg)

### 09/05

#### PCR

Fukuda

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 9 |
| 2 | M13F | M13R | 10 |
| 3 | 14-3-3 zeta F | 14-3-3 zeta R | 9 |
| 4 | 14-3-3 zeta F | 14-3-3 zeta R | 10 |
| 5 | tropomyosin F | tropomyosin R | 11 |
| 6 | tropomyosin F | tropomyosin R | 12 |

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

### 09/05

#### PCR

Fukuda

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | tropomyosin F | tropomyosin R | 11 |
| 2 | tropomyosin F | tropomyosin R | 11 |
| 3 | tropomyosin F | tropomyosin R | 11 |
| 4 | tropomyosin F | tropomyosin R | 12 |
| 5 | tropomyosin F | tropomyosin R | 12 |
| 6 | tropomyosin F | tropomyosin R | 12 |
| 7 | 14-3-3 pro F | 14-3-3 pro R | 13 |
| 8 | 14-3-3 pro F | 14-3-3 pro R | 13 |
| 9 | 14-3-3 pro F | 14-3-3 pro R | 13 |
| 10 | 14-3-3 pro F | 14-3-3 pro R | 14 |
| 11 | 14-3-3 pro F | 14-3-3 pro R | 14 |
| 12 | 14-3-3 pro F | 14-3-3 pro R | 14 |

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/a/ac/IGKU_b2_107_0.jpeg)

### 09/05

#### PCR

Fukuda

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | tropomyosin F | tropomyosin R | 11 |
| 2 | tropomyosin F | tropomyosin R | 11 |
| 3 | tropomyosin F | tropomyosin R | 11 |
| 4 | tropomyosin F | tropomyosin R | 12 |
| 5 | tropomyosin F | tropomyosin R | 12 |
| 6 | tropomyosin F | tropomyosin R | 12 |
| 7 | eef-1g F | eef-1g R | 4 |
| 8 | AK1 F | AK1 R | 5 |
| 9 | M13F | M13R | 9 |
| 10 | M13F | M13R | 9 |
| 11 | M13F | M13R | 9 |
| 12 | M13F | M13R | 9 |
| 13 | M13F | M13R | 10 |
| 14 | M13F | M13R | 10 |
| 15 | M13F | M13R | 10 |
| 16 | M13F | M13R | 10 |

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/c/cd/IGKU_b2_108_0.jpeg)

### 09/06

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | gfpp | 5.0 | EcoRI | 2.0 | None | None | H | 1.0 | 10.0 |
| 2 | gfpp | 5.0 | Not1 | 1.0 | HindⅢ | 1.0 | K | 1.0 | 10.0 |

![](https://static.igem.org/mediawiki/2017/b/be/IGKU_b2_098_0.jpeg)

### 09/06

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 163-1 |
| 2 | M13F | M13R | 163-2 |
| 3 | M13F | M13R | 164 |
| 4 | M13F | M13R | 142 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 20sec
61.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/2/2c/IGKU_b2_099_0.jpeg)

### 09/06

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK29 | IK24 | Rev+t |
| 2 | IK29 | IK24 | Rev+t |
| 3 | IK29 | IK24 | Rev+t |
| 4 | IK29 | IK24 | Rev+t |
| 5 | IK21 | IK24 | Rev+t, pro PCRp |
| 6 | IK29 | IK30 | Rev7(plasmid) |
| 7 | IK29 | IK30 | Rev7(plasmid) |
| 8 | IK29 | IK30 | Rev7(plasmid) |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/d/d8/IGKU_b2_102_0.jpeg)

### 09/06

#### PCR

Dou

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | P1 |
| 2 | IK21 | IK22 | P1 |
| 3 | IK21 | IK22 | P1 |
| 4 | IK21 | IK22 | P1 |

PCR Polymerase : 2X KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/9/9c/IGKU_b2_103_0.jpeg)

### 09/07

#### PCR

Dou

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | p+Rev |
| 2 | IK21 | IK22 | p+Rev |

PCR Polymerase : 2X KAPA Robust
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/7/7f/IGKU_b2_104_0.jpeg)

### 09/07

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | M13R | 142 PCR |
| 2 | IK60 | M13R | 142-1 |
| 3 | IK60 | M13R | 142-2 |
| 4 | IK60 | M13R | 142-3 |
| 5 | IK60 | M13R | 142-4 |
| 6 | IK60 | M13R | 142-5 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/e/ef/IGKU_b2_105_0.jpeg)

### 09/07

#### PCR

Fukuda

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK61 | IK62 | K733018 |
| 2 | IK61 | IK62 | K731250 |

PCR Polymerase : KAPA Robust
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 15sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/c/c8/IGKU_b2_110_0.jpeg)

### 09/07

#### PCR

Dou

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | P1 |
| 2 | IK21 | IK22 | P1 |
| 3 | IK21 | IK22 | P1 |

PCR Polymerase : 2X KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/f/f9/IGKU_b2_111_0.jpeg)

### 09/08

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 141 | 5.0 | XbaI | 1.0 | NotI | 0.5 | K | 1.0 | 10.0 |
| 2 | 144 | 5.0 | XbaI | 1.0 | NotI | 0.5 | K | 1.0 | 10.0 |
| 3 | 143 | 5.0 | XbaI | 1.0 | NotI | 0.5 | K | 1.0 | 10.0 |
| 4 | 145 | 5.0 | XbaI | 1.0 | NotI | 0.5 | K | 1.0 | 10.0 |

### 09/08

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK23 | IK28 | Rev3 |
| 2 | IK23 | IK28 | Rev4 |
| 3 | IK23 | IK28 | 2(nega) |
| 4 | IK23 | IK28 | Rev new |
| 5 | IK29 | IK30 | Rev3 |
| 6 | IK29 | IK30 | Rev4 |
| 7 | IK29 | IK30 | 2(nega) |
| 8 | IK29 | IK30 | Rev new |

PCR Polymerase : KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/5/54/IGKU_b2_113_0.jpeg)

### 09/08

#### PCR

Dou

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | p(blue font) |
| 2 | IK21 | IK22 | p(blue font) |
| 3 | IK21 | IK22 | p(blue font) |
| 4 | IK21 | IK22 | p(blue font) |

PCR Polymerase : 2X KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/7/76/IGKU_b2_114_0.jpeg)

### 09/08

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK23 | IK24 | 99(nega) |
| 2 | IK23 | IK24 | 101 |
| 3 | IK23 | IK24 | Rev3 |
| 4 | IK23 | IK24 | Rev4 |
| 5 | IK23 | IK24 | Rev5 |
| 6 | IK23 | IK24 | Rev6 |
| 7 | IK23 | IK24 | T(posi) |
| 8 | IK29 | IK30 | 99 |
| 9 | IK29 | IK30 | Rev5 |
| 10 | IK29 | IK30 | Rev6 |

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/f/f1/IGKU_b2_115_0.jpeg)

### 09/08

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK24 | all |
| 2 | IK21 | IK24 | all |
| 3 | IK21 | IK22 | promoter |
| 4 | IK21 | IK22 | promoter |
| 5 | IK21 | IK22 | promoter |
| 6 | IK21 | IK22 | promoter |
| 7 | IK29 | IK30 | Rev |
| 8 | IK29 | IK30 | Rev |
| 9 | IK29 | IK30 | nega 99 |
| 10 | IK29 | IK30 | water |
| 11 | IK29 | IK24 | Rev+t |
| 12 | IK29 | IK24 | Rev+t |
| 13 | IK23 | IK24 | plasmid |
| 14 | IK23 | IK24 | nega 99 |
| 15 | IK23 | IK24 | posi(T PCRp) |
| 16 | IK23 | IK24 | posi(T PCRp) |
| 17 | IK23 | IK24 | posi(T PCRp) |
| 18 | IK23 | IK24 | posi(T PCRp) |
| 19 | IK23 | IK24 | water |

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
55.0°C : 1min
72.0°C : 1min
72.0°C : 5min
10.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/b/b3/IGKU_b2_120_0.jpeg)

### 09/08

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | PCRp X10 |
| 2 | IK21 | IK22 | PCRp X10 |
| 3 | IK21 | IK22 | genome X10 |
| 4 | IK21 | IK22 | genome |
| 5 | IK21 | IK22 | water |
| 6 | IK21 | IK24 | all |
| 7 | IK21 | IK24 | p(PCRp),Rev+t gel ex |
| 8 | IK21 | IK24 | 8/20 PCRp,Rev+t gel ex |
| 9 | IK21 | IK24 | RA IK21X10 |
| 10 | IK21 | IK24 | RA |
| 11 | IK21 | IK24 | water |

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min
72.0°C : 5min
10.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/e/e8/IGKU_b2_121_0.jpeg)

### 09/09

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | 143 | 2 | 141.0 | 2.0 |
| 2 | 145 | 2 | 144.0 | 2.0 |
| 3 | 143 | 2 | 144.0 | 2.0 |

### 09/09

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 142 | 20.0 | XbaI | 4.0 | NotI | 2.0 | K | 4.0 | 40.0 |

### 09/09

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | 323 | 0.34 | GFPp(gal1) | 12.5 |

### 09/10

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 142-3 | 5.0 | XbaI | 0.5 | NotI | 0.5 | K | 1.0 | 10.0 |
| 2 | 142-5 | 5.0 | XbaI | 0.5 | NotI | 0.5 | K | 1.0 | 10.0 |
| 3 | 161-1 | 5.0 | XbaI | 0.5 | NotI | 0.5 | K | 1.0 | 10.0 |
| 4 | 163-1 | 5.0 | XbaI | 0.5 | NotI | 0.5 | K | 1.0 | 10.0 |
| 5 | 164 | 5.0 | XbaI | 0.5 | NotI | 0.5 | K | 1.0 | 10.0 |
| 6 | 166 | 5.0 | XbaI | 0.5 | NotI | 0.5 | K | 1.0 | 10.0 |

### 09/14

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | IK60 | 162 |
| 2 | IK29 | IK24 | Rev |
| 3 | IK40 | IK55 | GFP |

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
58.0°C : 1min
72.0°C : 1min
72.0°C : 2min
10.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/9/9e/IGKU_b2_124_0.jpeg)

### 09/14

#### Ligation

Yoshimoto

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | YEplac195/sma1 | 0.34 | 163.0 | 13.1 |
| 2 | YEplac195/sma1 | 0.34 | 166.0 | 17.25 |
| 3 | 143 | 2 | 141.0 | 2.0 |
| 4 | 145 | 2 | 144.0 | 2.0 |
| 5 | 143 | 2 | 144.0 | 2.0 |

### 09/14

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK21 | IK22 | genome |
| 2 | IK21 | IK22 | genome |
| 3 | IK121 | IK122 | genome |
| 4 | IK121 | IK122 | genome |
| 5 | IK129 | IK130 | Rev |
| 6 | IK129 | IK130 | Rev |
| 7 | IK129 | IK130 | nega 99 |
| 8 | IK129 | IK130 | water |
| 9 | IK21 | IK22 | water |
| 10 | IK121 | IK122 | water |

PCR Polymerase : 2X primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 15sec
72.0°C : 5sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle
PCR pulification: 1,2
PCR pulification: 4,5
Gel extraction: 1,2
Gel extraction: 4,5

![](https://static.igem.org/mediawiki/2017/5/5d/IGKU_b2_126_0.jpeg)

### 09/14

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK121 | IK122 | p |
| 2 | IK121 | IK122 | p |
| 3 | IK21 | IK22 | p |
| 4 | IK21 | IK22 | p |
| 5 | IK121 | IK122 | genome |
| 6 | IK121 | IK122 | genome |
| 7 | IK21 | IK24 | p1, Rev+t |
| 8 | IK21 | IK24 | p1, Rev+t |
| 9 | IK129 | IK24 | Rev new, t |
| 10 | IK21 | IK130 | p, Rev new |
| 11 | IK129 | IK24 | water |
| 12 | IK21 | IK130 | water |

PCR Polymerase : prime star max
98.0°C : 3min
98.0°C : 10sec
55.0°C : 15sec
72.0°C : 5sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/2/2d/IGKU_b2_127_0.jpeg)

### 09/15

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK129 | IK130 | Rev |
| 2 | IK129 | IK130 | Rev |
| 3 | IK129 | IK130 | Rev |
| 4 | IK21 | IK24 | OP2+(Rev+t) |
| 5 | IK129 | IK24 | NR1+t |
| 6 | IK21 | IK130 | NR1+OP2 |

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/9/99/IGKU_b2_128_0.jpeg)

### 09/15

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK129 | IK24 | NR1+t |
| 2 | IK129 | IK24 | NR1+t |
| 3 | IK129 | IK24 | NR1+t |
| 4 | IK129 | IK24 | NR1+t |
| 5 | IK129 | IK24 | water |
| 6 | K129 | IK130 | rev |
| 7 | K129 | IK130 | rev |

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~5
PCR pulification: 6,7
Gel extraction: 1~5
Gel extraction: 6,7

![](https://static.igem.org/mediawiki/2017/0/0e/IGKU_b2_129_0.jpeg)

### 09/15

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK129 | IK24 | RT1 |
| 2 | IK121 | IK122 | genome |
| 3 | IK121 | IK122 | genome |
| 4 | IK121 | IK122 | genome |
| 5 | IK21 | IK24 | RT1+OP1 |
| 6 | IK21 | IK24 | RT1+OP2 |
| 7 | IK121 | IK24 | RT1+NP1 |

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/6/6e/IGKU_b2_130_0.jpeg)

### 09/15

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK121 | IK24 | RT1+NP1 |
| 2 | IK121 | IK24 | RT1+NP1 |
| 3 | IK121 | IK24 | RT1+NP1 |
| 4 | IK121 | IK24 | RT1+NP1 |
| 5 | IK129 | IK24 | RT1 |
| 6 | IK121 | IK24 | water |

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~4
Gel extraction: 1~4

![](https://static.igem.org/mediawiki/2017/1/11/IGKU_b2_131_0.jpeg)

### 09/16

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | M13R | 161-1 |
| 2 | IK60 | M13R | 161-2 |
| 3 | IK60 | M13R | 163-1 |
| 4 | IK60 | M13R | 163-2 |
| 5 | IK60 | M13R | 163-3 |
| 6 | IK60 | M13R | 163-4 |
| 7 | IK60 | M13R | 166-1 |
| 8 | IK60 | M13R | 166-2 |
| 9 | IK60 | M13R | 166-3 |
| 10 | IK60 | M13R | 166-4 |
| 11 | IK60 | M13R | 166-5 |
| 12 | IK60 | M13R | 166-6 |
| 13 | IK60 | M13R | 162-1 |
| 14 | IK60 | M13R | 162-3 |
| 15 | IK60 | M13R | 161-1 |
| 16 | IK60 | M13R | 165-2 |
| 17 | IK60 | M13R | 165-3 |
| 18 | IK60 | M13R | 165-4 |
| 19 | IK60 | M13R | 165-5 |
| 20 | IK60 | M13R | 167-1 |
| 21 | IK60 | M13R | 167-2 |
| 22 | IK60 | M13R | 167-3 |
| 23 | M13R | IK40 | Gal1GFP 1 |
| 24 | M13R | IK40 | Gal1GFP 2 |
| 25 | M13R | IK40 | Gal1GFP 3 |
| 26 | M13R | IK40 | Gal1GFP 4 |

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 09/16

#### Ligation

Ohara

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | 325 | 0.528 | prt2 | 0.26 |
| 2 | 325 | 3.65 | prt2 | 7.33 |

![](https://static.igem.org/mediawiki/2017/8/81/IGKU_b2_133_0.jpeg)

### 09/16

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK121 | IK24 | prt2 |
| 2 | IK121 | IK24 | prt3 |
| 3 | IK121 | IK24 | prt3 |
| 4 | IK121 | IK24 | prt3 |
| 5 | IK121 | IK24 | prt3 |
| 6 | IK121 | IK122 | genome |

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle
PCR pulification: 2~5
Gel extraction: 2~5

![](https://static.igem.org/mediawiki/2017/1/16/IGKU_b2_134_0.jpeg)

### 09/16

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | M13R | 142-5 |
| 2 | IK60 | M13R | 142-5 |
| 3 | M13F | IK60 | 143-1 |
| 4 | M13F | IK60 | 143-2 |
| 5 | M13F | IK60 | 143-3 |
| 6 | M13F | IK60 | 145-1 |
| 7 | M13F | IK60 | 145-2 |
| 8 | M13F | IK60 | 145-3 |

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/6/61/IGKU_b2_135_0.jpeg)

### 09/16

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | genome |

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 09/16

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | M13F | 164-1 |
| 2 | IK60 | M13F | 164-2 |
| 3 | M13F | IK40 | Gal1 GFP 1 |
| 4 | M13F | IK40 | Gal1 GFP 2 |
| 5 | M13F | IK40 | Gal1 GFP 3 |
| 6 | M13F | IK40 | Gal1 GFP 4 |

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/1/1f/IGKU_b2_137_0.jpeg)

### 09/17

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK121' | IK24 | N(blue colony) |
| 2 | IK121' | IK24 | 1(Rev white) |
| 3 | IK121' | IK24 | 2 |
| 4 | IK121' | IK24 | 3 |
| 5 | IK121' | IK24 | prt3 |
| 6 | IK121' | IK122' | genome |

PCR Polymerase : prime star
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

### 09/17

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | 172-0 |
| 2 | M13F | M13R | 172-1 |
| 3 | M13F | M13R | 172-2 |
| 4 | M13F | M13R | 172-3 |
| 5 | M13F | M13R | 172-4 |
| 6 | M13F | M13R | 172-5 |
| 7 | M13F | M13R | 172-6 |
| 8 | M13F | M13R | 172-8 |
| 9 | M13F | M13R | 172-9 |
| 10 | M13F | M13R | 172-10 |
| 11 | M13F | M13R | 172-11 |
| 12 | M13F | M13R | 172-12 |
| 13 | M13F | M13R | 172-13 |
| 14 | M13F | M13R | 114-11 |
| 15 | M13F | M13R | 114-13 |
| 16 | M13F | M13R | 114-0 |
| 17 | M13F | M13R | 171-1-1 |
| 18 | M13F | M13R | 171-2 |
| 19 | M13F | M13R | 174-1 |
| 20 | M13F | M13R | 175-1 |
| 21 | M13F | M13R | 175-2 |
| 22 | M13F | M13R | 175-3 |
| 23 | M13F | M13R | 175-4 |
| 24 | M13F | M13R | 176-0 |
| 25 | M13F | M13R | 176-1 |
| 26 | M13F | M13R | 176-2 |
| 27 | M13F | M13R | 177-1 |
| 28 | M13F | M13R | 177-2 |
| 29 | M13F | M13R | 177-3 |
| 30 | M13F | M13R | 177-4 |
| 31 | M13F | M13R | 177-5 |
| 32 | M13F | M13R | water |
| 33 | M13F | M13R | 26(GPD AK1) |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/8/8e/IGKU_b2_140_0.jpeg)

### 09/17

#### Ligation

Ohara

| sample | Vector name | Vector amount(ul) | Insert name | Insert amount(ul) |
| --- | --- | --- | --- | --- |
| 1 | 325 | 0.528 | prt4 | 11.7 |

![](https://static.igem.org/mediawiki/2017/9/9c/IGKU_b2_141_0.jpeg)

### 09/18

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK40 | IK47 | 114-0 |
| 2 | IK55 | IK40 | 171-1 |
| 3 | IK40 | IK41 | 172-1 |
| 4 | IK60 | AK1F | 176-0 |
| 5 | IK60 | AK1F | 177-1 |
| 6 | IK60 | AK1F | 177-2 |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
58.0°C : 1min
72.0°C : 50sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 09/18

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | M13R | 104-1 |
| 2 | IK60 | M13R | 104-2 |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
60.0°C : 1min
72.0°C : 5min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/4/45/IGKU_b2_142_0.jpeg)

### 09/19

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | PrimerF | PrimerR | 162-1 |
| 2 | PrimerF | PrimerR | 162-1 |
| 3 | PrimerF | PrimerR | 162-1 |
| 4 | PrimerF | PrimerR | 162-1 |
| 5 | PrimerF | PrimerR | Rev |
| 6 | PrimerF | PrimerR | Rev |
| 7 | PrimerF | PrimerR | Rev |
| 8 | PrimerF | PrimerR | Rev |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/5/5f/IGKU_b2_143_0.jpeg)

### 09/19

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | Ik29 | IK30 | OR1-1 |
| 2 | Ik29 | IK30 | OR1-2 |
| 3 | Ik29 | IK30 | OR1-3 |
| 4 | Ik29 | IK30 | OR1-4 |
| 5 | Ik29 | IK30 | OR1 nega |
| 6 | Ik29 | IK30 | R2 |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/c/c4/IGKU_b2_144_0.jpeg)

### 09/19

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK60 | M13R | 162-1 |
| 2 | IK60 | M13R | 162-2 |
| 3 | IK60 | M13R | 162-3 |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/1/1d/IGKU_b2_145_0.jpeg)

### 09/19

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK89 | IK90 | EGFP |
| 2 | IK89 | IK90 | EGFP |
| 3 | IK84 | IK85 | REV |
| 4 | IK86 | IK87 | REV |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane1,2
PCR pulification: lane3
Gel extraction: lane1,2
Gel extraction: lane3

![](https://static.igem.org/mediawiki/2017/a/a9/IGKU_b2_146_0.jpeg)

### 09/20

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK40 | IK55 | 171 |
| 2 | IK40 | IK55 | 171 |
| 3 | IK40 | IK55 | 171 |
| 4 | IK40 | IK55 | 171 |
| 5 | IK40 | IK55 | 171 |
| 6 | IK40 | IK55 | 171 |
| 7 | IK40 | IK60 | 177 |
| 8 | IK40 | IK60 | 177 |
| 9 | IK40 | IK41 | 172 |
| 10 | IK40 | IK41 | 172 |
| 11 | IK40 | IK41 | 172 |
| 12 | IK40 | IK41 | 172 |
| 13 | IK40 | IK41 | 172 |
| 14 | IK40 | IK41 | 172 |
| 15 | IK40 | IK60 | 176 |
| 16 | IK40 | IK60 | 176 |
| 17 | IK46 | M13F | 148 |
| 18 | IK46 | M13F | 148 |
| 19 | IK46 | M13F | 148 |
| 20 | IK46 | M13F | 148 |
| 21 | IK46 | M13F | 148 |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/a/ac/IGKU_b2_148_0.jpeg)![](https://static.igem.org/mediawiki/2017/1/18/IGKU_b2_148_1.jpeg)

### 09/21

#### PCR

Yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | OR-1(REV plasmid) |
| 2 | M13F | M13R | OR-2 |
| 3 | M13F | M13R | OR-3 |
| 4 | M13F | M13R | OR-4 |
| 5 | M13F | M13R | OR-5 |
| 6 | IK23 | Ik24 | OR-1 |
| 7 | IK23 | Ik24 | OR-2 |
| 8 | IK23 | Ik24 | OR-3 |
| 9 | IK23 | Ik24 | OR-4 |
| 10 | IK23 | Ik24 | OR-5 |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

![](https://static.igem.org/mediawiki/2017/c/c5/IGKU_b2_149_0.jpeg)

### 09/22

#### PCR

Ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK121 | IK24 | prt3 |
| 2 | IK121 | IK24 | num99 |
| 3 | IK121 | IK24 | OR1-1 |
| 4 | IK121 | IK24 | OR1-2 |
| 5 | IK121 | IK24 | OR1-3 |
| 6 | IK121 | IK24 | OR1-5 |
| 7 | IK121 | IK24 | OR1-4 |

PCR Polymerase : prime star max
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle![](https://static.igem.org/mediawiki/2017/b/be/IGKU_b2_150_0.jpeg)

![](https://static.igem.org/mediawiki/2017/b/be/IGKU_b2_150_0.jpeg)

### 09/22

#### PCR

Dou

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK89 | IK90 | EGFP2-1 |
| 2 | IK89 | IK90 | EGFP2-2 |

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/04

#### Restriction Enzyme digestion

yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Template 5 | 5.0 | Xba | 0.025 | EcoR1 | 0.025 | M | 1.0 | 10.0 |
| 2 | Template 5 | 5.0 | Not1 | 0.025 | EcoR1 | 0.025 | H | 1.0 | 10.0 |
| 3 | Template 5 | 5.0 | Not1 | 0.025 | Xba1 | 0.025 | H | 1.0 | 10.0 |

### 10/04

#### PCR

ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD21 | OD23 | eGFP plasmid |
| 2 | OD21 | OD23 | eGFP plasmid |
| 3 | OD22 | OD23 | eGFP plasmid |
| 4 | OD22 | OD23 | eGFP plasmid |
| 5 | OD31 | OD32 | T7-RRE c |
| 6 | OD31 | OD32 | T7-RRE c |

PCR Polymerase : prime star max
98.0°C : 10sec
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 10sec
15.0°C : Infinate
X35 cycle

### 10/04

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | pSB1C3 | 15.0 | Xba1 | 5.0 | Spe1 | 5.0 | M | 12.0 | 100.0 |
| 2 | pSB1C3 | 15.0 | Xba1 | 5.0 | Pst1 | 5.0 | M | 12.0 | 100.0 |

### 10/05

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | vf | vr | Colony1~20(191) |
| 2 | vf | vr | Colony21~40(192) |
| 3 | vf | vr | Colony41~60(193) |
| 4 | vf | vr | Colony61~80(194) |

PCR Polymerase : prime star max
98.0°C : 10sec
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 10sec
15.0°C : Infinate
X35 cycle

### 10/05

#### Restriction Enzyme digestion

yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | template | 5.0 | Pst1 | 0.05 | 10xH | 1.0 | 10.0 |

### 10/06

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD41 | OD42 | Gal-GFP |
| 2 | OD41 | OD43 | Gal-GFP |
| 3 | OD41 | OD44 | Gal-GFP |
| 4 | IK61 | OD52 | Gal-GFP |
| 5 | OD53 | OD54 | Gal-GFP |

PCR Polymerase : prime star max
98.0°C : 10sec
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 10sec
15.0°C : Infinate
X35 cycle

### 10/07

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK61 | OD54 | T7-RRE |
| 2 | IK61 | OD54 | fukuda GFP |

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X40 cycle

### 10/07

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD11 | OD12 | GPD-gfp |
| 2 | OD11 | OD13 | GPD-gfp |
| 3 | OD41 | OD42 | Gal-GFP |
| 4 | OD41 | OD43 | Gal-GFP |
| 5 | OD41 | OD44 | Gal-GFP |
| 6 | OD31 | OD32 | T7-RRE |
| 7 | IK61 | IK54 | fukuda GFP + T7RRE |
| 8 | IK61 | IK54 | fukuda GFP + T7RRE |

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X40 cycle

### 10/09

#### Restriction Enzyme digestion

yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | RE2 name | RE2 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | T7 RRE | 15.0 | Xba1 | 3.0 | Pst1 | 3.0 | M | 3.0 | 30.0 |

### 10/09

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK61 | IK54 | GFP+RRE |

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X40 cycle

### 10/09

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD41 | OD42 | Gal-gfp |
| 2 | OD41 | OD44 | Gal-gfp |

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X35 cycle

### 10/12

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD61 | OD12 | TDH3-eGFP |
| 2 | OD62 | OD12 | TDH3-eGFP |
| 3 | OD63 | OD12 | TDH3-eGFP |
| 4 | OD61 | OD13 | TDH3-eGFP |
| 5 | OD62 | OD13 | TDH3-eGFP |
| 6 | OD63 | OD13 | TDH3-eGFP |
| 7 | OD73 | IK62 | GFP |
| 8 | OD45 | OD42 | GFP |
| 9 | OD45 | OD43 | GFP |
| 10 | OD45 | OD44 | GFP |
| 11 | IK61 | OD74 | GFP |

PCR Polymerase : KAPA HiFi fast
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/12

#### Restriction Enzyme digestion

Yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Template14 | 5.0 | Xba1 | 1.0 | M | 1.0 | 10.0 |
| 2 | Template100 | 5.0 | Xba1 | 1.0 | M | 1.0 | 10.0 |

### 10/12

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD62 | OD12 | yeast genome |
| 2 | OD62 | OD13 | yeast genome |
| 3 | OD73 | IK62 | fukuda gfp |
| 4 | OD45 | OD42 | gal gfp |
| 5 | IK61 | OD74 | fukuda gfp |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/13

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD62 | OD12 | yeast genome |
| 2 | OD62 | OD12 | yeast genome |
| 3 | OD62 | OD13 | TDH3-eGFP |
| 4 | OD62 | OD13 | TDH3-eGFP |
| 5 | OD73 | IK62 | fukuda GFP |
| 6 | IK61 | OD71 | fukuda GFP |
| 7 | OD72 | OD54 | T7-RRE |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/14

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD62 | OD12 | yeast genome |
| 2 | OD62 | OD13 | yeast genome |
| 3 | OD45 | OD42 | gal1 GFP |
| 4 | IK61 | OD74 | fukuda GFP |
| 5 | OD73 | IK62 | fukuda GFP |

PCR Polymerase : KAPA
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/14

#### PCR

ohara

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK103 | IK104 | 165 gBlock |
| 2 | IK107 | IK108 | 161 gBlock |
| 3 | IK101 | IK102 | 161 gBlock |
| 4 | IK105 | IK106 | 163 gBlock |

PCR Polymerase : yoshimoto
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/14

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD45 | OD42 | gal1 GFP |
| 2 | IK61 | OD74 | fukuda GFP |
| 3 | OD73 | IK62 | fukuda GFP |
| 4 | IK61 | OD54 | fukuda GFP |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/14

#### Restriction Enzyme digestion

yoshimoto

| lane | Sample DNA name | DNA amount (ul) | RE1 name | RE1 amount(ul) | Buffer name | Buffer amount(ul) | Total volume(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | template | 15.0 | Xba1 | 3.0 | M | 3.0 | 100.0 |
| 2 | template | 15.0 | Xba1 | 5.0 | M | 3.0 | 100.0 |

### 10/14

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD31 | OD32 | T7+RRE |
| 2 | OD22 | OD23 | eGFP |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/15

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK61 | OD54 | 6+7 |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/15

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK85 | IK86 | rev |
| 2 | IK87 | Ik88 | rev |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~2
Gel extraction: 1~2

### 10/15

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD31 | OD32 | T7+RRE |
| 2 | OD22 | OD23 | TDH3promoter |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/15

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD31 | OD32 | T7+RRE |
| 2 | OD22 | OD23 | TDH3promoter |
| 3 | OD45 | OD42 | Gal eGFP |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/15

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK105 | Ik106 | Gblock 163 |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/15

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK105 | IK108 | Gal1 loop |
| 2 | IK101 | Ik104 | GFP loop |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

### 10/15

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK85 | IK86 | Rev parts |
| 2 | IK87 | IK88 | Rev parts |
| 3 | IK61 | OD54 | T7-RRE-GFP |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/17

#### PCR

yoshitomo

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK61 | OD54 | 4 |
| 2 | IK61 | OD54 | 5 |
| 3 | IK61 | OD54 | 7 |

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/17

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK87 | IK88 | Rev |
| 2 | IK105 | IK108 | Gal1-loop |

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
55.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/18

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK105 | IK108 | template1 |

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/18

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | IK107 | IK108 | Gal1pro-loop |
| 2 | IK105 | IK106 | Gal1pro-loop |

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/19

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD22 | OD23 | GPD-loop |
| 2 | IK87 | IK88 | Gal1-loop |
| 3 | IK105 | IK108 | T7-RRE |
| 4 | IK101 | IK104 | TDH3-eGFP |
| 5 | OD31 | OD32 | Rev |

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/20

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | M13F | M13R | Colony 1-10 (RRE) |
| 2 | M13F | M13R | Colony11-17 (TDH3-eGFP) |
| 3 | M13F | M13R | Colony 18-21 (Rev) |
| 4 | M13F | M13R | Colony 22-23 (GPDp-loop) |
| 5 | M13F | M13R | Colony24-28 (GPDp-loop) |
| 6 | M13F | M13R | Colony 29-32 (T7-RRE) |
| 7 | M13F | M13R | Colony 33-35 (TDH3-eGFP) |
| 8 | M13F | M13R | Colony 36-37 (GPDp-loop) |
| 9 | M13F | M13R | Colony 38-40 (Gal1-loop) |
| 10 | M13F | M13R | Colony 41-43 (Rev) |
| 11 | M13F | M13R | Colony 44 (GPDp-loop) |

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

### 10/22

#### PCR

yoshimoto

| lane | Primer Foward | Primer Revese | Template |
| --- | --- | --- | --- |
| 1 | OD22 | OD23 | TDH3 GFP |
| 2 | IK87 | IK88 | Rev |
| 3 | IK105 | IK108 | Gal1 |
| 4 | IK101 | IK104 | GPD1 |
| 5 | OD31 | OD32 | T7-RRE |

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Kyoto/Experiments

* [![](https://static.igem.org/mediawiki/2017/4/4a/Kyototitle222.png)](https://2017.igem.org/Team%3AKyoto)
* ![](https://static.igem.org/mediawiki/2017/6/6e/松５.png)
* [Project](https://2017.igem.org/team%3AKyoto/Project)
  + [Description](https://2017.igem.org/team%3AKyoto/Project)
  + [Results](https://2017.igem.org/Team%3AKyoto/Results)
  + [Demonstrate](https://2017.igem.org/Team%3AKyoto/Demonstrate)
  + [Discussion](https://2017.igem.org/Team%3AKyoto/Discussion)
  + [Basic Parts](https://2017.igem.org/Team%3AKyoto/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3AKyoto/Composite_Part)
* [Practices](https://2017.igem.org/Team%3AKyoto/Human_Practices)
  + [Human Practices](https://2017.igem.org/Team%3AKyoto/Human_Practices)
  + [Integrated Practices](https://2017.igem.org/Team%3AKyoto/Integrated_Practices)
  + [Public Engagement](https://2017.igem.org/Team%3AKyoto/Engagement)
  + [Collaborations](https://2017.igem.org/Team%3AKyoto/Collaborations)
  + [Events](https://2017.igem.org/Team%3AKyoto/Events)
  + [Further Consideration](https://2017.igem.org/Team%3AKyoto/Discussion_HP)
* [Experiment](https://2017.igem.org/Team%3AKyoto/Notebook)
  + [Notebook](https://2017.igem.org/Team%3AKyoto/Notebook)
  + [Material&Methods](https://2017.igem.org/Team%3AKyoto/Experiments)
* [Team](https://2017.igem.org/Team%3AKyoto/Team)
  + [Members](https://2017.igem.org/Team%3AKyoto/Team)
  + [Attributions](https://2017.igem.org/Team%3AKyoto/Attributions)
* [Criteria](https://2017.igem.org/Team%3AKyoto/Achievements)
  + [Achievements](https://2017.igem.org/Team%3AKyoto/Achievements)
  + [HP/Silver](https://2017.igem.org/Team%3AKyoto/HP/Silver)
  + [HP/Gold](https://2017.igem.org/Team%3AKyoto/HP/Gold_Integrated)

[![](https://static.igem.org/mediawiki/2017/c/c5/Kyoto_notebook_jump.png)](#wrapper)

# Material&Methods

* [1) Parts](#Parts)
* [2) Primer list](#Primer)
* [3) Materials](#Materials)

+ [3-1 Kit](#Kit)
+ [3-2 Restriction Enzyme](#Restriction Enzyme)
+ [3-3 Polymerase](#Polymerase)
+ [3-4 DNA ligase](#DNA ligase)
+ [3-5 Marker](#Marker)
+ [3-6 Organism](#Organism)
+ [3-7 Antibiotics](#Antibiotics)
+ [3-8 Equipment](#Equipment)
+ [3-9 Backbone](#Backbone)
+ [3-10 Buffer](#Buffer)
+ [3-11 SDS-PAGE&Westernblotting](#SDS-PAGE&Westernblotting)

* [4) Methods](#methods)

+ [4-1 Miniprep](#Miniprep)
+ [4-2 Gel Extraction and PCR purification](#Gel Extraction and PCR purification)
+ [4-3 Restriction Enzyme Digestion](#Restriction Enzyme Digestion)
+ [4-4 Ligation](#Ligation)
+ [4-5 Transformation](#Transformation)
+ [4-6 PCR](#PCR)
+ [4-7 Sequencing](#Sequencing)
+ [4-8 RT-PCR](#RT-PCR)
+ [4-9 RTqPCR](#RTqPCR)
+ [4-10 Western blotting (Sample preparation of *S. cerevisiae*)](#Western blotting (Sample preparation of S. cerevisiae))
+ [4-11 Western blotting (Basic protocol)](#Western blotting (Basic protocol))
+ [4-12 Culture using yeasts and measurement](#Culture using yeasts and measurement)
+ [4-13 The way of taking photos](#The way of taking photos)
+ [4-14 Methods of taking movies](#Methods of taking movies)
+ [4-15 Way of making agar pad](#Way of making agar pad)
+ [4-16 Synthesis of dsRNA](#dsRNA)
+ [4-17 Soaking](#Soaking)

##### 1) Parts

###### [Basic Parts](https://2017.igem.org/Team%3AKyoto/Basic_Part)

###### [Composite Parts](https://2017.igem.org/Team%3AKyoto/Composite_Part)

##### 2) Primer List

| primer name | sequence | Length | Tm | GC% | Designer | Manufacturer |
| --- | --- | --- | --- | --- | --- | --- |
| tropomyosinF | GATCGAGAAGGACAACGCCC | 20 | 65 | 60 | Fukuda | Macrogen Japan Corp |
| tropomyosinT7R | TAATACGACTCACTATAGGGCTTGTTTTCTCCGGCTTCGG | 40 | 79 | 48 | Fukuda | Macrogen Japan Corp |
| tropomyosinT7F | TAATACGACTCACTATAGGGGATCGAGAAGGACAACGCCC | 40 | 79 | 50 | Fukuda | Macrogen Japan Corp |
| tropomyosinR | CTTGTTTTCTCCGGCTTCGG | 20 | 66 | 55 | Fukuda | Macrogen Japan Corp |
| top-1F | GACTTTTCGTGCGTCTTCGG | 20 | 64 | 55 | Fukuda | Macrogen Japan Corp |
| top-1T7R | TAATACGACTCACTATAGGGCCCGGTTGAAAAGCAAGTGT | 40 | 78 | 45 | Fukuda | Macrogen Japan Corp |
| top-1T7F | TAATACGACTCACTATAGGGGACTTTTCGTGCGTCTTCGG | 40 | 78 | 48 | Fukuda | Macrogen Japan Corp |
| top-1R | CCCGGTTGAAAAGCAAGTGT | 20 | 64 | 50 | Fukuda | Macrogen Japan Corp |
| eef-1gF | GCAAAGGTTGCCGGTAAGAC | 20 | 64 | 55 | Fukuda | Macrogen Japan Corp |
| eef-1gT7R | TAATACGACTCACTATAGGGGACGGCAGAGGCCAACTTTA | 40 | 78 | 48 | Fukuda | Macrogen Japan Corp |
| eef-1gT7F | TAA TACGACTCACTATAGGGGCAAAGGTTGCCGGTAAGAC | 40 | 77 | 48 | Fukuda | Macrogen Japan Corp |
| eef-1gR | GACGGCAGAGGCCAACTTTA | 20 | 64 | 55 | Fukuda | Macrogen Japan Corp |
| asbF | GGACTTTGGTCGTTGTCCCA | 20 | 63 | 55 | Fukuda | Macrogen Japan Corp |
| asbT7R | TAATACGACTCACTATAGGGTCCACAGCGTCCTTCAAGTC | 40 | 75 | 48 | Fukuda | Macrogen Japan Corp |
| asbT7F | TAATACGACTCACTATAGGGGGACTTTGGTCGTTGTCCCA | 40 | 78 | 48 | Fukuda | Macrogen Japan Corp |
| asbR | TCCACAGCGTCCTTCAAGTC | 20 | 61 | 55 | Fukuda | Macrogen Japan Corp |
| 14-3-3proteinF-2 | TGGAGGGTGATCTCGTCCAT | 20 | 62 | 55 | Fukuda | Macrogen Japan Corp |
| 14-3-3proteinT7R-2 | TAATACGACTCACTATAGGG CAAGGGGGATGGTTGGTCTC | 41 | 78 | 49 | Fukuda | Macrogen Japan Corp |
| 14-3-3proteinT7F-2 | TAATACGACTCACTATAGGG TGGAGGGTGATCTCGTCCAT | 41 | 76 | 46 | Fukuda | Macrogen Japan Corp |
| 14-3-3proteinR-2 | CAAGGGGGATGGTTGGTCTC | 20 | 65 | 60 | Fukuda | Macrogen Japan Corp |
| actinF | AATGGCTCCGGTATGTGCAA | 20 | 65 | 50 | Fukuda | Macrogen Japan Corp |
| actinR | TGGTGGTGAAAGAGTAGCCG | 20 | 62 | 55 | Fukuda | Macrogen Japan Corp |
| 14-3-3zetaF | CCTACAAGAACGTGGTCGGT | 20 | 61 | 55 | Fukuda | Macrogen Japan Corp |
| 14-3-3zetaT7R | TAATACGACTCACTATAGGGTGTGAGTGCTCGCAAACTGT | 40 | 75 | 45 | Fukuda | Macrogen Japan Corp |
| 14-3-3zetaT7F | TAATACGACTCACTATAGGGCCTACAAGAACGTGGTCGGT | 40 | 76 | 48 | Fukuda | Macrogen Japan Corp |
| 14-3-3zetaR | TGTGAGTGCTCGCAAACTGT | 20 | 60 | 50 | Fukuda | Macrogen Japan Corp |
| 14-3-3proF | TCTCGGTCGCCTACAAGAAC | 20 | 61 | 55 | Fukuda | Macrogen Japan Corp |
| 14-3-3proT7R | TAATACGACTCACTATAGGGCCAGTCTCTCCCGTCTCGTT | 40 | 77 | 50 | Fukuda | Macrogen Japan Corp |
| 14-3-3proT7F | TAATACGACTCACTATAGGGTCTCGGTCGCCTACAAGAAC | 40 | 75 | 48 | Fukuda | Macrogen Japan Corp |
| 14-3-3proR | CCAGTCTCTCCCGTCTCGTT | 20 | 62 | 60 | Fukuda | Macrogen Japan Corp |
| AK1F | TCGGAGACGGTTGTGTGAAGATG | 23 | 66 | 52 | Yoshimoto | Macrogen Japan Corp |
| AK1T7R | TAATACGACTCACTATAGGGCAAGCACGGGTTGAATGGGT | 41 | 79 | 46 | Yoshimoto | Macrogen Japan Corp |
| AK1T7F | TAATACGACTCACTATAGGGTCGGAGACGGTTGTGTGAAGATG | 43 | 77 | 47 | Yoshimoto | Macrogen Japan Corp |
| AK1R | CAAGCACGGGTTGAATGGGT | 20 | 66 | 55 | Yoshimoto | Macrogen Japan Corp |
| longAK2F | TCCGAATCATGGCGAGAACC | 20 | 66 | 55 | Yoshimoto | Macrogen Japan Corp |
| longAK2T7R | TAATACGACTCACTATAGGGCATGCCGGTCAACGGATAGT | 40 | 78 | 48 | Yoshimoto | Macrogen Japan Corp |
| longAK2T7F | TAATACGACTCACTATAGGGTCCGAATCATGGCGAGAACC | 40 | 78 | 48 | Yoshimoto | Macrogen Japan Corp |
| longAK2R | CATGCCGGTCAACGGATAGT | 20 | 64 | 55 | Yoshimoto | Macrogen Japan Corp |
| shortAK2F | CCGTTTTGGATTTGTCGCGT | 20 | 67 | 50 | Yoshimoto | Macrogen Japan Corp |
| shortAK2T7R | TAATACGACTCACTATAGGGTCGATCTTCTTGACCGTCGC | 40 | 77 | 48 | Yoshimoto | Macrogen Japan Corp |
| shortAK2T7F | TAATACGACTCACTATAGGGCCGTTTTGGATTTGTCGCGT | 40 | 79 | 45 | Yoshimoto | Macrogen Japan Corp |
| shortAK2R | TCGATCTTCTTGACCGTCGC | 20 | 64 | 55 | Yoshimoto | Macrogen Japan Corp |
| gal1-1F | cccCGAATTCccccattatctt | 22 | 68 | 50 | Yoshimoto | Macrogen Japan Corp |
| gal1-1R | CTCCCACACCAGCATCCAA | 19 | 63 | 58 | Yoshimoto | Macrogen Japan Corp |
| gal1-2F | AAGCTGGGCGCCACTTT | 17 | 64 | 59 | Yoshimoto | Macrogen Japan Corp |
| gal1-2R | CAAGTCGTTGCTGAAGAAACATTTCAC | 27 | 66 | 41 | Yoshimoto | Macrogen Japan Corp |
| gal1-3F | ACTCGGCGTCCGGAG | 15 | 61 | 73 | Yoshimoto | Macrogen Japan Corp |
| gal1-3R | tacccTGCAGgggagc | 16 | 59 | 69 | Yoshimoto | Macrogen Japan Corp |
| gpd-1F | tctagaggatccccCGAATTC | 21 | 63 | 52 | Yoshimoto | Macrogen Japan Corp |
| gpd-1R | CCGTTGACCGGCATGct | 17 | 65 | 65 | Yoshimoto | Macrogen Japan Corp |
| gpd-2F | AATCCGCTGTGGCCGC | 16 | 66 | 69 | Yoshimoto | Macrogen Japan Corp |
| gpd-2R | AACCACCTTCTTCGCACAGAAC | 22 | 63 | 50 | Yoshimoto | Macrogen Japan Corp |
| gpd-3F | GACGTCCTTGGTGAAATGTTTC | 22 | 61 | 45 | Yoshimoto | Macrogen Japan Corp |
| gpd-3R | gggaaaaccctggcgttac | 19 | 64 | 58 | Yoshimoto | Macrogen Japan Corp |
| IK21 | CCTCCACAAAGGCCTCTCCT | 20 | 64 | 60 | Yoshimoto | Macrogen Japan Corp |
| IK22 | CTTTATATTATCAATATTTGTGTTTGTGGAGGGGG | 35 | 70 | 34 | Yoshimoto | Macrogen Japan Corp |
| IK23 | ATTTATTGGAGAAAGATAACATATCATACTTTCCC | 35 | 66 | 29 | Yoshimoto | Macrogen Japan Corp |
| IK24 | AACCTTTATTGTGTGCACACTCAAAC | 26 | 63 | 38 | Yoshimoto | Macrogen Japan Corp |
| IK25 | GATAATATAAAGATGGGCAGCAGCCATCAT | 30 | 69 | 40 | Yoshimoto | Macrogen Japan Corp |
| IK26 | CCAATAAATCTATTCTTTAGCTCCTGACTCCAATATTGTA | 40 | 70 | 32 | Yoshimoto | Macrogen Japan Corp |
| IK27 | ACTAGTGGATCCCCCCCTCCACAAAGGCCTCTCCT | 35 | 81 | 60 | Yoshimoto | Macrogen Japan Corp |
| IK28 | GAATTCCTGCAGCCCAACCTTTATTGTGTGCACACTCAAAC | 41 | 80 | 46 | Yoshimoto | Macrogen Japan Corp |
| IK60 | GTGTAGGCCTCGGCATCCG | 19 | 67 | 68 | Yoshimoto | Macrogen Japan Corp |
| IK61 | TAATACGACTCACTATAGGGGTGGAGAGGGTGAAGGTGATG | 41 | 77 | 49 | Yoshimoto | Macrogen Japan Corp |
| IK62 | TAATACGACTCACTATAGGGTGCCATGTGTAATCCCAGCAG | 41 | 77 | 46 | Yoshimoto | Macrogen Japan Corp |
| IK81 | atgtatTCTAGAcTCCTCCACAAAGGCCTCTCCTGG | 36 | 75 | 50 | Dou | Macrogen Japan Corp |
| IK82 | GCCCCCTGCAtGCCCTCC | 18 | 72 | 78 | Dou | Macrogen Japan Corp |
| IK83 | GCGCAGGAGGGCaTGCAGG | 19 | 73 | 74 | Dou | Macrogen Japan Corp |
| IK84 | gtACTAGTgCTTTATATTATCAATATTTGTGTTTGTGGAGGGGGGGG | 47 | 77 | 40 | Dou | Macrogen Japan Corp |
| IK85 | GGGTCGCGGATCCgaaCtcATGG | 23 | 75 | 65 | Dou | Macrogen Japan Corp |
| IK86 | CGCTTCTTCCTGCCATgaGttcGGA | 25 | 74 | 56 | Dou | Macrogen Japan Corp |
| IK87 | atgtatTCTAGAcaatgggcagcagccatc | 30 | 71 | 47 | Dou | Macrogen Japan Corp |
| IK88 | gtACTAGTgCTATTCTTTAGCTCCTGACTCCA | 32 | 66 | 44 | Dou | Macrogen Japan Corp |
| IK89 | AAT CTA GAA GCG GCC GCA CAC GCT TT TTC | 38 | 77 | 39 | Dou | Macrogen Japan Corp |
| IK90 | GAACTAGTCAGAGATCTGAGTCCGGACTTGTACAGCTC | 38 | 73 | 50 | Dou | Macrogen Japan Corp |
| IK91 | ccgcaaaatcaggtgtgttg | 20 | 63 | 50 | Yoshimoto | Macrogen Japan Corp |
| IK92 | tatctttaccaattagtttcaatgtttagtaagg | 34 | 63 | 26 | Yoshimoto | Macrogen Japan Corp |
| IK93 | AATGACGCTGACGGTACAGG | 20 | 61 | 55 | Yoshimoto | Macrogen Japan Corp |
| IK94 | ATGCCCCAGACTGTGAGTTG | 20 | 61 | 55 | Yoshimoto | Macrogen Japan Corp |
| IK95 | ggttgtgtgaagatgaccgc | 20 | 61 | 55 | Yoshimoto | Macrogen Japan Corp |
| IK96 | tcttcagcagggacttgcag | 20 | 62 | 55 | Yoshimoto | Macrogen Japan Corp |
| IK97 | agcgttcaactagcagacca | 20 | 59 | 50 | Yoshimoto | Macrogen Japan Corp |
| IK98 | agggcagattgtgtggacag | 20 | 61 | 55 | Yoshimoto | Macrogen Japan Corp |
| IK99 | TGGCGAGAACCACCTTCTTC | 20 | 63 | 55 | Yoshimoto | Macrogen Japan Corp |
| IK100 | GCTCCCTGGTTGAACGTGTA | 21 | 61 | 52 | Yoshimoto | Macrogen Japan Corp |
| IK21-2 | CCTCCACAAAGGCCTCTCCT | 20 | 64 | 60 | Yoshimoto | Macrogen Japan Corp |
| IK22-2 | CTTTATATTATCAATATTTGTGTTTGTGGAGGGGG | 35 | 70 | 34 | Yoshimoto | Macrogen Japan Corp |
| IK121 | GCCTCTCCTGGGGTTTGAG | 19 | 63 | 63 | Ohara | Macrogen Japan Corp |
| IK122 | CTCCACAAACACAAATATTGATAATATAAAGatgggcagc | 40 | 73 | 35 | Ohara | Macrogen Japan Corp |
| IK129 | CCTCCACAAACACAAATATTGATAATATAAAGatgggcagcagccatcat | 50 | 80 | 38 | Ohara | Macrogen Japan Corp |
| IK130 | GGAAAGTATGATATGTTATCTTTCTCCAATAAATCTATTCTTTAGCTCCTGACTCCAATATTGTA | 65 | 77 | 31 | Ohara | Macrogen Japan Corp |
| IK101 | atgtcttctagagcggtaggt | 21 | 56 | 48 | Yoshimoto | Macrogen Japan Corp |
| IK102 | cctgattttgcggccgcatccgtcgaaactaagttctgg | 39 | 85 | 54 | Yoshimoto | Macrogen Japan Corp |
| IK103 | aacttagtttcgacggat gcggccgcaaaatcag g | 36 | 83 | 50 | Yoshimoto | Macrogen Japan Corp |
| IK104 | gcACTAGTtgaagcttctctatct | 24 | 56 | 42 | Yoshimoto | Macrogen Japan Corp |
| IK105 | atgtcttctagagccccattatcttagcctaaaaaaacc | 39 | 73 | 38 | Yoshimoto | Macrogen Japan Corp |
| IK106 | cctgattttgcggccgcctccatggtggcggc | 32 | 90 | 69 | Yoshimoto | Macrogen Japan Corp |
| IK107 | cccgccgccaccatggaggcggccgcaaaatcagg | 35 | 94 | 71 | Yoshimoto | Macrogen Japan Corp |

##### 3) Materials

###### 3-1 Kit

| Name | Supplier |
| --- | --- |
| Wizard® SV Gel and PCR | Promega |
| FastGene™Plasmid Mini Kit | NIPPON Genetics Co.,Ltd |

###### 3-2 Restriction Enzyme

| Name | Supplier |
| --- | --- |
| EcoRI | TaKaRa, Promega |
| PstI | TaKaRa, Promega |
| SpeI | TaKaRa, Promega |

###### 3-3 Polymerase

| Name | Supplier |
| --- | --- |
| KAPA™HiFi HotStart ReadyMix (2x) | KAPABIOSYSTEMS |
| KAPA2G™ Fast HotStart ReadyMix with dye (2x) | KAPABIOSYSTEMS |
| KAPATaq™EXtra HotStart ReadyMix with dye | KAPABIOSYSTEMS |

###### 3-4 DNA ligase

| Name | Supplier |
| --- | --- |
| T4 DNA ligase | TaKaRa |

###### 3-5 Marker

| Name | Supplier |
| --- | --- |
| 1kb DNA Ladder | TaKaRa |

###### 3-6 Organism

| Name | Supplier |
| --- | --- |
| E.coli DH5α Genotype | TaKaRa |
| E.coli BL21(DE3)pLysS Competent Cells | Promega |

###### 3-7 Antibiotics

| Name | Supplier |
| --- | --- |
| Chloramphenicol | Wako |
| Ampicillin | Wako |

###### 3-8 Equipment

| Name | Supplier |
| --- | --- |
| BioPhotometer | eppendorf |
| LABO SHAKER | BIO CRAFT |
| CO2 INCUBATOR | SANYO |
| Pipette Controller | Biohit Midi Plus |
| MiniCentrifuge Model GMC-060 | LMS CO.,LTD. |
| HIGH-SPEED REFRIGERATED CENTRIFUGE | TOMY |
| HIGH-PRESSURE STEAM STERILIZER | TOMY |
| VOTEX-GENE2 | Scientific Industryes |
| Scanning Electron Miniscope TM1000s | HITACHI |
| Fluorescence Microscope BX61N-34-FL-1-D | OLYMPUS |
| SCIECE IMAGING SYSTEM LAS-3000 | Fuji film |

###### 3-9 Backbone

| Name | Supplier |
| --- | --- |
| pSB1C3 | iGEM registry |
| pSB1A2 | iGEM registry |
| pSB3C5 | iGEM registry |

###### 3-10 Buffer

| Name | Supplier |
| --- | --- |
| Sodium Carbonate | Wako |
| Sodium Bicarbonate | Wako |
| Methanol(99.5%) | Wako |
| Sodium Chloride | Wako |
| SDS | nacalai tesque |
| Trisaminomethane | nacalai tesque |
| Potassium dihydrogenphosphste | SIGAMA-ALDRICH |
| Potassium Chloride | Wako |
| Glycine | Wako |
| Agar, Powder | Wako |
| Agarose XP | Wako |
| 10% Hydrochloric Acid | Wako |

###### 3-11 SDS-PAGE&Westernblotting

| IMMOBILON - P Blotting Sandwiches | Immobilon |
| --- | --- |
| REAL GEL PLATE (10%) | BIO CRAFT |
| BE-210(SDS-PAGE) | BIO CRAFT |
| BE-330 | BIO CRAFT |
| Amersham ECL Anti-Mouse IgG | GE healthcare |
| Amersham ECL Anti-rabbit IgG | GE healthcare |
| Amersham ECL Prime Blocking Reagent | GE healthcare |
| Amersham ECL Prime WB Detection Reagent | GE healthcare |

|
|  |
|
|
|
|
|
|
|

##### 4) Methods

###### 4-1 Miniprep

Minipreps were performed using FastGene™Plasmid Mini Kit and and Wizard® Plus SV Minipreps DNA Purification System according to the manufacturer's protocols.

###### 4-2 Gel Extraction and PCR purification

Gel Extraction was performed using Wizard® SV Gel and PCR Clean-Up System according to the manufacturer's protocols.

###### 4-3 Restriction Enzyme Digestion

Restriction enzyme treatment was performed using Takara Bio's or NEB's restriction enzymes according to the respective manufacturer's protocols.

###### 4-4 Ligation

Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's

###### 4-5 Transformation

1. Thaw competent cells on ice
2. Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes
3. Keep them in heating block for 45 seconds, then cool on ice for 2 minutes
4. Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37°C
5. Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37°C

###### 4-6 PCR

PCR was performed using KAPA™HiFi HotStart ReadyMix (2x), KAPA™ 2G Robust HotStart ReadyMix (2x), PrimeSTAR® Max DNA Polymerase, GoTaq®, Takara Ex Taq, and Quick Taq® HS DyeMix according to the manufacturer's protocols

###### 4-7 Sequencing

We outsourced the sequencing to Macrogen

http://www.macrogen-japan.co.jp/cap\_seq\_0203.php

###### 4-8 RT-PCR

We extracted *B. xylophilus* whole mRNA using Sepazol®-RNA Ⅰ Super G according to the manufacturer's protocol.

RT-PCR was performed using ~~~ according to the manufacturer's protocol.

###### 4-9 qRT-PCR

We inserted 3 plasmids written on the lower left into 2 types of yeasts written on the lower right.

|  |  |
| --- | --- |
| p14 Gal1p-AK1  p26 GPDp-AK1  p100 GPF-GFP | MKY13 WT  MKY117 ski2Δ |

Regarding p14, we conducted

1. Create culture with raffinose medium as primary culture.
2. Dilute it in glucose medium and galactose medium and incubate for 12-15 hours.
3. Collect at log phase of OD = 1.1- 1.8 (MKY13) 0.6 - 1.2 .(MKY117)
4. Collect RNA by Lucigen's MasterPure Yeast RNA purification kit. (http://www.lucigen.com/home.php?cat=273)
5. Do DNase treatment according to the manual for 20 minutes.
6. Dissolve the obtained total RNA in 15 μL and dilute it to prepare a standard curve sample (using p14 sample and p100 sample). The measurement sample was diluted 5000 times with water and used. Analysis is ABI Step-one plus. SuperScript III Platinum SYBR qRT-PCR kit (https://www.thermofisher.com/order/catalog/product/11732020) was used.

The three following primers were used :

* IK91-IK92: This amplifies the loop region of hairpin RNA
* IK95-IK96: This amplifies the inside of AK1 mRNA
* Kota 030 - Kota 031: It amplifies near the 5 'end of 25S rRNA. (Fujii Kitabatake et al., 2009)

Quantitation was performed with n = 3 biological triplicate, and the variation in RNA recovery amount was standardized by dividing the quantified value of loop and the quantified value of AK1 by the quantitative value of 25S rRNA, respectively.

###### 4-10 Western blotting (Sample preparation of *S. cerevisiae*)

1. Cultivate 1ml of *S. cerevisiae* culture whose OD600 values are 1.0
2. Pour 1ml of the *S. cerevisiae* cell culture into 1.5ml tubes
3. Centrifuge 5000rpm for 1min and remove the supernatant
4. Resuspend with 30ul of SDS sample buffer
5. Heat samples for 10min at 100°C using block incubater
6. Vortex well

###### 4-11 Western blotting (Basic protocol)

1. Apply sample to SDS-PAGE minigel (BIOCRAFT 15%)
2. Soak the gel in transfer buffer
3. Soak PVDF membrane in 100% methanol for 30 sec
4. Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min
5. Set filter paper, membrane, gels, filter paper in this order to semidry transfer from anode to cathode
6. Transfer the proteins from the gel to the membrane with 100 mA for 1 h
7. Soak membrane in blocking buffer (dilution of 5 g ECL Blocking reagent by 100ml of TBST) for 1 h
8. Wash for 5 min 3 times with TBST
9. Apply 1' antibody (dilution of 10 μl of 1’antibody by 10ml of Blocking buffer) and shake for 1 h
10. Wash for 5 min 3 times with TBST
11. Apply 2' antibody (dilution of 4μl of 2’antibody by 10ml of Blocking buffer) and shake for 1 h
12. Wash for 5 min 3 times with TBST
13. Drain excess wash buffer from the washed membrane and place on flat surface, protein side up
14. Add detection reagent onto the membrane, covering all of the membrane
15. Incubate for 5 minutes at room temperature
16. Drain off excess detection reagent by dabbing with Kimwipe
17. Place the sample in the CCD camera compartment and record the images

###### 4-12 Culture using yeasts and measurement

1. Prepare medium put on 3.5 cm plate.
2. Drop liquid-cultured yeasts on the center of medium.
3. Disperse them by bacteria spreader and dry for 30min to 1 hour. Or leave them as they are.
4. Pipette suspension including nematodes on the medium.
5. Culture it at 25 ℃

###### 4-13 The way of taking photos

1. Culture yeasts expressing GFP over night and dilute them three times with DW or SD liquid medium.
2. Drop 200-300μl of water on the medium after one or two days, and pipette it in order to spread over the medium.
3. Suck out 12ul of water on the medium and drop it on microscope slide.
4. Cover it with cover glass and observe whether nematodes feed yeasts expressing GFP by fluorescence microscope.

###### 4-14 Methods of taking movies

1. Make agar pad.
2. Extract nematodes from PDA medium, collect them into 1.5ml tube and centrifuge it.
3. Remove supernatant and put 1.5ul of suspension with nematodes into gap of cover glasses.
4. Snap microscope slide with the finger and reach suspension to the center of agarose medium.
5. Observe whether nematodes feed yeast expressing GFP by microscope and take videos of it.

###### 4-15 Way of making agar pad 1. Make medium of 2% agar and sterlize by autoclave. 2. Put sterlized slide glass between two of slide glasses covered with "Kimwipes", drop agar medium, then rapidly cover agar medium with another slide glass. 3. After the medium sodifies, reveal slowly covered slide glass. 4. Pipette yeasts on the medium, cover the medium on cover glass, and culture it for 1-2days. 4-16 Synthesis of dsRNA We synthesyzed dsRNA by "MEGAscript™ T7 Transcription Kit." <https://www.thermofisher.com/order/catalog/product/AM1334> 4-17 Soaking We followed this paper as for soaking. [https://link.springer.com/content/pdf/10.1007%2Fs10658-012-0035-0.pdf](https://link.springer.com/content/pdf/10.1007/s10658-012-0035-0.pdf)

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# Team:Kyoto/Results

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# Results

##### Table of contents

* [1) Observe that *B. xylophilus* feeds on yeast](#res1)
* [2) Identify *B. xylophilus* which feeds yeast by fluorescence](#res2)
* [3) Choose dsRNA](#res3)
* [4) Conduct feeding RNAi in yeast](#res4)
* [5) Observe that *B. xylophilus* feeds yeast expressing dsRNA](#res5)
* [6) Improve transition of mRNA to cytosol](#res6)

##### 1) Observe that *B. xylophilus* feeds on yeast

Dr. Y Takeuchi, a researcher of *B. xylophilus*, gave us a strain that was bred in the laboratory. These nematodes were cultivated with gray mold. It was also possible to grow nematodes on the plate growing gray mold.

After we washed nematodes cultured in mold with lactic acid to remove mold, we placed them with budding yeast on a new plate. At first we observed nematodes by microscope, but we could not observe them eating yeast. Although they tried to pierce yeast with their stylet, it seemed to be difficult for them to penetrate it well because the yeast was not fixed on the plate then moved, and also originally the yeast was too small.

So we thought it would be easier to pierce with a stylet if the yeast was bigger, and we modified the conditions and used diploid yeast instead of haploid yeast, which had been used thus far. Visually, diploid yeast is different from haploid with respect to shape and size, where diploid is bigger than haploid (fig 1-a). Therefore, we decided to continue observation with diploid yeast.

![](https://static.igem.org/mediawiki/2017/3/3d/Kyoto_fig1a.jpeg)

**Figure 1-a** *S. cerevisiae* cells.
Diploid (light blue) and haploid (orange) cells were mixed and photographed to compare their sizes.

We also improved the filming conditions. We stopped observing the nematodes on the medium directly, and changed to a method of sandwiching the medium and yeast between the slide glass and the cover glass and injecting nematodes from the side. By doing so, the yeast was fixed firmly on the medium and we could solve the problem that the yeast moved when nematodes were trying to pierce it with their stylet.

Shown in the movie below is the moment *B. xylophilus* preyed on budding yeast (Movie 1). This was filmed for the first time in the world. The needle-shaped structure found near the nematode's mouth is a stylet. The nematode pushed the stylet against the yeast and inserted it in the yeast. The yeast started to shrink rapidly sometime after the nematode pierced it, and in the end its shape almost disappeared. Yeast has a robust cell wall, but it seems to be crushed with a strong force.

[](https://static.igem.org/mediawiki/2017/e/e9/B.xylophilus.mp4)
Movie 1 The moment *B. xylophilus* prey on *S. cerevisiae*.

The results of experiments on whether or not *B. xylophilus* that ate *S. cerevisiae* would grow was shown in the figure (Figure 1-b).

![](https://static.igem.org/mediawiki/2017/1/16/Kyoto_fig1b.png)

**Figure 1-b** Survival rate of *B. xylophilus* with different foods.
Diploid or haploid cells were grown in SD liquid culture and spread on agar plates. 100 nematodes were grown on each plate and the number of survivors were counted at the indicated time points. (n=3)

In the case of haploid or no food, the survival rate decreases at Day 4, but when using diploids the survival rate increases. The large cell diploid yeast seems easier to ingest for *B. xylophilus*.

##### 2) Identify *B. xylophilus* which ate yeast by fluorescence

In order to confirm the effect of RNAi, we needed a marker to distinguish nematodes that preyed on yeast from the others within the experimental period. Cultured nematodes included many growth stages, so it was anticipated that they included some nematodes at the stages where they could not eat yeast. In order to distinguish only the individuals which ate yeast and identify the effect, we decided to repeat the feeding experiment using yeast which fluoresces with eGFP.

The results of observation of nematodes which ate yeast with a fluorescence microscope are shown in the figure (Figure 2-a~Figure 2-d).

The intestines of *B. xylophilus* were highlighted by green fluorescence penetrating through the center throughout the entire body. The mouth is on the left side and the anus is on the right side. eGFP seemed to be very stable in the intestine, and the entire intestine fluoresced uniformly. There was some fluorescence discontinued in the center, and this was correspondent with the part where *B. xylophilus* gonads crossed over the intestine.

From the above results, it was found that by expressing eGFP, it became possible to clearly distinguish nematodes that preyed on yeast from the others. By expressing dsRNA simultaneously with eGFP, it should be possible to measure the effect of dsRNA efficiently. As can be seen from this photograph, feeding nematodes on yeast expressing GFP also made it possible to clearly observe the structure of *B. xylophilus* intestine. This method seemed to be effective for closely observing abnormalities occurring in an intestine of *B. xylophilus*, like gastroscopy using barium in humans.

![](https://static.igem.org/mediawiki/2017/2/27/Kyoto_fig2a2b.png)

![](https://static.igem.org/mediawiki/2017/b/b6/Kyoto_fig2c2d.png)

**Figure 2-a** eGFP expression cassette.
**Figure 2-b** Yeast cells expressing eGFP.
eGFP expression cassette was cloned into a plasmid and introduced into wild type *S. cerevisiae* strain.
Images were recorded by fluorescence microscopy.
**Figure 2-c** Nematodes with GFP signal.
Nematodes fed on Yeast were visualized by fluorescence microscopy. Fed on eGFP(+) yeast (left) and EGFP(-) yeast (right) are shown. Scale bar: 50μm
**Figure 2-d** Nematodes with GFP signal 2.
(top) Nematode with GFP fluorescence was inspected by confocal microscopy. XY plane and Z plane (cross section) are shown, respectively.
(bottom) Negative control. Yeast cells with no eGFP plasmid were used. Scale bar: 25μm

Among the nematodes being cultured, how many nematodes were thought to surely eat yeast? We collected nematodes on the 3rd and 7th day from the start of the culture by eGFP yeast and observed what proportion of them were fluorescent using a fluorescence microscope.
The results are shown in the figure (Figure 2-e).

In observation immediately after giving yeast, nematodes emitting fluorescence could not be observed, but about 30% of nematodes emitted fluorescence at Day 3. From this, it was found that at least 30% of nematodes at day 3 were ingesting yeast by this time (Figure 2-e). Interestingly, keeping the plate warm as it was continued observation, it was observed that the number of fluorescent nematodes was reduced to about half (15% of the total) in Day 7. (p = 0.001) Yeasts applied to nutrient-free water agar may die due to starvation or dryness by this time, and the supply of new GFP to nematodes has stopped, which may be the cause of this decrease. The fact that the proportion of fluorescent nematodes declines means that GFP in the intestine of nematodes is digested and degraded over time.

![](https://static.igem.org/mediawiki/2017/8/8e/Flo2.png)

**Figure 2-e** Time course of the rate of eGFP(+) nematodes.
Nematodes were grown on eGFP(+) yeast and examined by fluorescence microscopy at the indicated time. (n=18)

##### 3) Choose dsRNA

In order to kill *B. xylophilus*, it is necessary to efficiently knock-down genes essential for growth. Examining the literature, we found a paper that succeeded in knocking down essential genes and reducing the survival rate by submerging *B. xylophilus* in high concentrations of dsRNA for a certain period of time (soaking RNAi) [1]. In this paper, the target was mRNA of arginine kinase AK1 which was an essential gene expressed in the intestines and RNAi of AK1 showed a fatal effect also in C. elegans. AK1 is an invertebrate-specific key enzyme of energy metabolism so it is often used as a target for development of invertebrate-specific inhibitors. It was a promising candidate for dsRNA expressed in yeast.
In order to select the most ideal target, we prepared dsRNA of several target mRNAs including AK1 in vitro and tried soaking RNAi. We obtained target sequence from a public database, designed oligos, and cloned genes by RT-PCR. At this time, we put the T7 promoter on both ends of the DNA so that dsRNA was synthesized by in vitro transcription. After transcription, association of dsRNA was induced by an annealing operation, and the template was removed with DNase. We confirmed the dsRNA finally obtained by electrophoresis (Figure 3-a).

![](https://static.igem.org/mediawiki/2017/7/79/Kyoto_fig3a.png)

**Figure 3-a** In vitro synthesized dsRNAs for soaking RNAi experiments.
M DNA size marker, λSty I
1 dsAK-2 (692-bp)
2 dsEef-1g (528-bp)
3 dsAK-1 (449-bp)
4 dsAsb (559-bp)
5 ds14-3-3zeta (534-bp)
6 dsTropomyosin (532-bp)
7 ds14-3-3 protein (610-bp)
8 dsGFP (649-bp)

We prepared these RNAs, adjusted to a concentration of 2 μg /μL, and tried soaking RNAi The results is shown in the figure (Figure 3-b). As is clearly shown, we could not see the phenotype due to the introduction of dsRNA which was inconsistent with the previously reported example. As a result of contacting several *B. xylophilus* researchers and gathering information, it turned out that even several Japanese researchers have attempted to reproduce *B. xylophilus* soaking RNAi, but no group was able to observe a clear effect. The reason may be that soaking RNAi of *B. xylophilus* contains technically unstable steps. Alternatively, since *B. xylophilus* used this time is derived from wild nematodes collected from the field, there may be a difference between the strain we used and nematodes in the publication where soaking RNAi was effective.

Although the effect of soaking RNAi was not observed, we decided to target the AK1 gene because there is already the report[1], and constructed the expression system of dsRNA in yeast.

![](https://static.igem.org/mediawiki/2017/8/83/Kyoto_fig3b.png)

**Figure 3-b** Effects of soaking RNAi
*B. xylophilus* were soaked into 2 mg/mL dsRNAs shown in Figure 3-a. After 4h incubation, nematodes were washed and incubated on M9 buffer plate (time=0h). Plates were examined for mortality of the nematodes up to 24h. The method of soaking RNAi was based on reference[1].

##### 4) Conduct feeding RNAi in yeast

In order to express AK1-dsRNA, we placed inverted repeat derived from AK1 ORF downstream of the Gal1 promoter of the part ([BBa\_K517000](http://parts.igem.org/wiki/index.php/Part%3ABBa_K517000)), and inserted a small loop sequence of 67-nt between repeats. There was a report that this loop sequence was effective when *S. cerevisiae* expressed long dsRNA[2]. Since *S. cerevisiae* has no Dicer homolog, dsRNA is not processed into siRNA. However, overexpression of dsRNA may be toxic to *S. cerevisiae*, so we adopted the Gal1 conditional promoter. When *S.cerevisiae* is cultured in the presence of glucose, this promoter is inactive, and many mRNAs are expressed when the carbon source of the medium is replaced with galactose. At the same time, we also used the GPD promoter ([BBa\_K517001](http://parts.igem.org/Part%3ABBa_K517001)) which is a constitutive expression type promoter. dsGFP with a sequence specific to GFP and was designed as a negative control. Outline of construction is shown below (Figure 4-a).

![](https://static.igem.org/mediawiki/2017/2/28/%E3%82%A2%E3%82%BB%E3%83%83%E3%83%88_14%404x.png)

**Figure 4-a** dsRNA expression vectors we used.
Left: Design of each dsRNA cassette. Plus strand and minus strand are tandemly transcribed as a single strand RNA.
 To enhance dsRNA formation, a short hairpin loop sequence was inserted in between.
Right: Our plasmids used in this study. Gal1 promoter or GPD promoter was fused to dsAK1 or dsGFP (negative control) cassettes. The cassetes were cloned in YEPlac195 plasmid (2-micron, high-copy number plasmid).

We cultured plasmid-containing yeasts in several media, collected RNA, and quantified by qRT-PCR with the "loop" part as a target.Moreover, it is known that various viruses of dsRNA type exist in *S. cerevisiae*. As a factor closely related to the life cycle of such a virus, Ski gene group is known. Many of these are now revealing detailed functions. The Ski complex binds to the 3 'end of RNA and serves as a cofactor for RNA exosome, which is an exonuclease complex that degrades RNA in the 3-5 direction. By binding to the 3 'end to disband the higher-order structure of RNA, it makes the recognition of substrate by exosome efficient. Since the dsRNA virus is known to proliferate in the ski2Δ strain [3], it was hoped that the use of this strain would greatly increase the yield of the target dsRNA.

The following is the result of qRT-PCR (Figure 4-c). First, expression of dsRNA was successfully detected when wild-type yeast into which Gal1 promoter-dsAK1 was introduced was induced by galactose. Almost the same values ​​are obtained even when the target of the primer set used for qRT-PCR is set to the loop portion or set within the AK1 gene. On the other hand, expression was suppressed as expected when replacing the medium with Glucose. From this, it was demonstrated that it is possible to conditionally induce long hairpin RNA expression using our plasmid.
Interestingly, the expression in Ski2Δ strain is higher than that in WT strain (p <0.05). This indicates that in wild type yeast, Ski complex is degrading targeting foreign dsRNA in addition to RNA virus as expected. From these results, it was found that it is possible to raise the intracellular concentration of exogenous dsRNA by using yeast mutant strain.
In GPD promoter ([BBa\_K517001](http://parts.igem.org/Part%3ABBa_K517001)), dsRNA could not be expressed. This part is composed of only the 112 bp sequence near the center out of the TDH 3 promoter (588 bp). Strong expression was confirmed when the 588 bp full-length promoter ([BBa\_K 530008](http://parts.igem.org/Part%3ABBa_K530008)) was used for eGFP expression experiments (Figure 2-a, 2-b), so we believe that there is a high probability that this part is defective.

![](https://static.igem.org/mediawiki/2017/1/12/Kyoto_fig4b.png)

**Figure 4-b** Quantification of dsRNA in *S. cerevisiae*
Dr. Makoto Kitabatake (Institute for frontier life and medical science, Kyoto University) performed qRT-PCR using purified yeast total RNAs. The indicated strains were grown in SD-Glucose or SD-Galactose and harvested at the mid to late log phase. Total RNAs were isolated by MasterPure Yeast RNA purification kit (Lucigen) and analyzed by SuperScript III Platinum SYBR qRT-PCR kit (Thermo) after DNase treatment. The primers used in this assay are shown. Quantification of dsRNA was normalized by 25S rRNA. (n=3)

##### 5) Observe that *B. xylophilus* feeds on yeast expressing dsRNA

We let *B. xylophilus* prey on the yeast prepared as described above and recorded the survival rate and behavior of nematodes as follows.

We counted the number of surviving nematodes which fed on dsRNA / eGFP expressing yeast every other day. We also confirmed the survival rate among nematodes that showed fluorescence of eGFP.

![](https://static.igem.org/mediawiki/2017/9/9c/Kyoto_fig5abc.png)

**Figure 5-a** Survival rate of *B. xylophilus* fed by yeast expressing dsAK1
Diploid yeast strain carrying the both dsAK1 and eGFP plasmids were grown in SD-Glucose (glu) or SD-Galactose (gal) medium and spread on new plates. ~100 nematodes were grown on each plate and examined by microscopy. (n=3)

**Figure 5-b** Mortality of GFP(+) nematodes
Nematodes fed by diploid yeast carrying dsAK1 and eGFP plasmids were examied by fluorescence microscopy. Gal, yeast culture was prepared by SD-Galactose medium (dsRNA induction). Glu, the same yeast strain was grown in SD-Glucose medium (dsRNA repression). (n=3)

**Figure 5-c** Survival rate of *B. xylophilus* fed by Ski2Δ strain
Feeding RNAi experiments were performed as in Figure 5-a. Ski2Δ strain instead of WT strain was used as prey. (n=1)

From the above results, the number of nematodes did not decline predominantly even when using yeast whose expression of dsAK1 was confirmed (Figure 5-a). According to previous experiments, when the nematodes cultured by eGFP(+) yeast, the rate of eGFP(+) labbeled nematodes was only about 30% (Figure 2-d). For this reason, even if dsRNA taken in kills nematodes, since the proportion of nematodes ingesting a sufficient number of yeasts to obtain the effect is not so high, there is a possibility that the effect given by dsRNA has been diluted. We focused only on nematodes that fed yeast and confirmed the mortality of fluorescent nematodes to evaluate the effect of dsRNA (Figure 5-b).
Even in this case, we could not confirm the effect of dsRNA as expected.
Moreover, the survival rate of nematodes is lower when using yeast cultured in +glu SD medium which should suppress the expression of dsRNA.
The results were the same even when using ski2Δ yeast in which the expression level of dsRNA was increased (Figure 5-c).
This seemingly contradictory result will be discussed later in the discussion.
We thought that there might be some obstacle before dsRNA was taken up by nematodes and sought out the cause.

##### 6) Improve transport of mRNA to cytosol

As shown in the figure (Figure 6-a), the diameter of the stylet is very small, about a fraction of a single cell of yeast. For this reason, *B. xylophilus* seemed to draw out the cytoplasmic fraction, but large cellular componentns such as the nucleus may not be efficiently consumed by *B. xylophilus.*

![](https://static.igem.org/mediawiki/2017/f/fd/Stylet.jpeg)

**Figure 6-a** Nematode’s stylet, diploid yeast, and haploid yeast. Scale bar : 10μm

A number of studies have been done on the nuclear export of mRNA, and the basic mechanism has been elucidated. It is known that various RNAs such as mRNA, rRNA, tRNA, etc. are recognized by transporters specific to each type and pass through the nuclear pore complex[4]. However, since the dsRNA as prepared this time does not exist in nature, it is not known whether there is a transport factor that recognizes this RNA or whether it is efficiently transported out of nucleus.

In order to prepare remedies for this problem, we tried experiments utilizing the REV factor of HIV-1 RNA, which is known to have the function of improving the efficiency of nuclear export of RNA.

As shown in the figure, REV plays the role of carrying an unspliced RNA genome to the cytoplasm in the life cycle of HIV-1 (Figure 6-b). In the case of ordinary mRNA, there is a retention mechanism that prevent molecules retaining introns from transferring out of the nucleus, thus preventing the transport of immature mRNA. REV binds to a specific part (RRE: Rev responsive element) of the intron on the HIV-1 RNA genome and binds itself to the nuclear export factor CRM1, and overcomes such a retention mechanism and transports RNA to the cytoplasm[5]. Even if the dsRNA is not recognized as a nuclear export factor or even if it is retained in the nuclear retention factor, we thought that it is possible that the efficiency of nuclear export can be improved by inserting REV-RRE system, and provide these new parts to the iGEM community ([BBa\_K2403000](http://parts.igem.org/Part%3ABBa_K2403000),
 [BBa\_K2403002](http://parts.igem.org/Part%3ABBa_K2403002))

![](https://static.igem.org/mediawiki/2017/d/de/Kyoto_fig6b.jpeg)

**Figure 6-b** Rev protein induces nuclear export of RRE-containing RNAs.

The results of microinjection of RI-labeled dsRNA with RRE into the nucleus of Xenopus oocytes are shown in the figure (Figure6c ~ Figure6f). Nuclear and cytoplasm were separated after a certain period of time following injection, RNA was recovered from each and analyzed .

![](https://static.igem.org/mediawiki/2017/8/86/Kyoto_fig6c.png)

![](https://static.igem.org/mediawiki/2017/7/75/Kyoto_fig6d.png)

![](https://static.igem.org/mediawiki/2017/6/6f/Kyoto_fig6fe.png)

\* Dr. Taniguchi, Institute for fronter life and medical science, Kyoto university, conducted an experiment on the process of treating RI on and after in vitro transcription.

**Figure 6-c** Outline of Xenopus oocyte microinjection

**Figure 6-d** RNAs produced by in vitro transcription
U6 and U6-RRE are used as controls.

**Figure 6-e** Microinjection of dsGFP with or without RRE into Xenopus oocyte
Indicated RNAs with or without Rev protein were injected into Xenopus oocyte nucleus. The oocytes were dissected at the indicated time points (t=0, 60 min). The nuclear RNAs (N) and the cytoplasmic RNAs (C) were extracted and analyzed by PAGE. The left pannel shows dsGFP with RRE (GFP-RRE + GFPrev), the right pannel shows dsGFP without RRE (GFPfwd + GFPrev).

**Figure 6-f** Long exposure

As expected, when U6-RRE is injected together with buffer without Rev, U6-RRE remains in the nucleus, whereas U6 - RRE is injected with buffer containing Rev, U6-RRE was remarkably transported outside the nucleus. This result demonstrated that nuclear export of RNA is promoted depending on both the RRE sequence and the Rev protein in the case of U6 RNA originally staying in the nucleus. These effects indicate that these parts are promising as devices for efficiently transporting highly structured RNA, which is often used in synthetic biology, to the cytoplasm.([BBa\_K2403000](http://parts.igem.org/Part%3ABBa_K2403000)
[BBa\_K2403002](http://parts.igem.org/Part%3ABBa_K2403002))
Unfortunately, transcription products of GFP-RRE were too thin to understand whether they responded to Rev. However, from the figure on the right of Figure 6-f, it was also found that dsRNA remained in the nucleus a lot. It is suggested that implementation of a system to promote nucleocytoplasmic transport is effective.
Compared to the signal at T = 0, since many signals are lost at 60 minutes after injection, there may be a mechanism for degrading dsRNA in cells. In addition to promoting transportation efficiency, there will be room for improvement to improve stability.

This is the Result obtained in this project.
We would like to discuss Discussion on interpretation of Result and Future plan.

###### Reference

* [1] X. rong Wang, X. Cheng, Y. dong Li, J. ai Zhang, Z. fen Zhang, and H. rong Wu, “Cloning arginine kinase gene and its RNAi in *Bursaphelenchus xylophilus* causing pine wilt disease,” Eur. J. Plant Pathol., vol. 134, no. 3, pp. 521–532, 2012.* * [2] A. Sigova, N. Rhind, and P. D. Zamore, “A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe,” genes Dev., 2004.
    * [3] R. Esteban and R. B. Wickner, “A new non-mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains.,” Genetics, 1987.
    * [4] M. T. B. Sloan, Katherine E, Pierre-Emmanuel Gleizes, “Nucleocytoplasmic Transport of RNAs and RNA–Protein Complexes,” J. Mol. Biol., vol. 428, no. 10, pp. 2040–2059, 2016.
    * [5] V. W. Pollard and M. H. Malim, “the Hiv-1 Rev Protein,” Annu. Rev. Microbiol., vol. 52, no. 1, pp. 491–532, 1998.

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