iGEM 2013 — Team OU-Norman_OK

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# Team:OU-Norman OK/Project/Notebook

### From 2013.igem.org

[![home](https://static.igem.org/mediawiki/2013/e/ef/Logo_small.png)](https://2013.igem.org/Team%3AOU-Norman_OK/Home)

* [Home](https://2013.igem.org/Team%3AOU-Norman_OK/Home)
* [Team](https://2013.igem.org/Team%3AOU-Norman_OK/Team)
  + [Undergraduates](https://2013.igem.org/Team%3AOU-Norman_OK/Team/Undergraduates)
  + [Graduate Students](https://2013.igem.org/Team%3AOU-Norman_OK/Team/Graduate_Students)
  + [Advisors](https://2013.igem.org/Team%3AOU-Norman_OK/Team/Advisors)
* [Project](https://2013.igem.org/Team%3AOU-Norman_OK/Project)
  + [Notebook](https://2013.igem.org/Team%3AOU-Norman_OK/Project/Notebook)
  + [Submitted Parts](https://2013.igem.org/Team%3AOU-Norman_OK/Project/Submitted_Parts)
* [Safety](https://2013.igem.org/Team%3AOU-Norman_OK/Safety)
* [Attributions](https://2013.igem.org/Team%3AOU-Norman_OK/Attributions)

#

09/13/13

**Gel of Pptb-**

| Row 1 || Lane Number | Sample || 1 | Ladder (1kb plus) || 2 | Pptb- #9A || 3 | Pptb- #9 B || 4 | Empty || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

[Back to top](#top)

#

09/13/13

**Digest of Pptb- Sample**

500ng X (μL/178.1ng) = 2.81μL

500ng X (μL/110.1ng) = 4.54μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || Pptb- #9A | 2μL | 2μL | 2μL | 2.81μL | 11.19μL | 20μL || Pptb- #9B | 2μL | 2μL | 2μL | 4.54μL | 9.46μL | 20μL ||

[Back to top](#top)

#

09/11/13

**Quantification and DNA Extraction of 3A**

QIAprep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/d/d6/92.png)

[Back to top](#top)

#

09/10/13

**Gel of Pptb- + Clos Ori**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Pptb- #1 || 3 | pptb- #2 || 4 | Pptb- #3 || 5 | Pptb- #4 || 6 | Pptb- #6 || 7 | Empty || 8 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/e/ec/91.png)

Expected insert size of 125bp

No valid inserts found

[Back to top](#top)

#

09/11/13

**Digest of Pptb-**

Pptb- #1

500ng X (μL/81.1ng) = 6.17μL

Pptb- #2

500ng X (μL/86.4ng) = 5.79μL

Pptb- #3

500ng X (μL/70.9ng) = 7.05μL

Pptb- #4

500ng X (μL/147.7ng) = 3.39μL

Pptb- #6

500ng X (μL/169.3ng) = 2.95μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || Pptb- #1 | 2μL | 2μL | 2μL | 6.17μL | 9.83μL | 20μL || Pptb- #2 | 2μL | 2μL | 2μL | 5.79μL | 8.21μL | 20μL || Pptb- #3 | 2μL | 2μL | 2μL | 7.05μL | 6.95μL | 20μL || Pptb- #4 | 2μL | 2μL | 2μL | 3.39μL | 10.61μL | 20μL || Pptb- #5 | 2μL | 2μL | 2μL | 2.95μL | 11.05μL | 20μL ||

[Back to top](#top)

#

09/11/13

**Quantification and DNA Extraction of 3A**

QIAprep Spin Miniprep Kit (250)
[Back to top](#top)

#

09/10/13

**Gel of Pptb- + Clos Ori**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Pptb- #1 || 3 | pptb- #2 || 4 | Pptb- #3 || 5 | Pptb- #4 || 6 | Pptb- #5 || 7 | Pptb- #7 || 8 | Pptb- #14 || 9 | Pptb- #15 || 10 | Empty || 11 | Empty || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || Row #2 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Pptb- #16 || 3 | Pptb- #17 || 4 | Pptb- #18 || 5 | Pptb- #21 || 6 | Pptb- #22 || 7 | Pptb- #23 || 8 | Pptb- #24 || 9 | Pptb- #25 || 10 | Empty || 11 | Empty || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || | | | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

[Back to top](#top)

#

09/6/13

**Digest of Pptb- and Clos Ori**

3 #17

500ng X (μL/123.2ng) = 4.06μL

Pptb- #1

500ng X (μL/104.6ng) = 4.78μL

Pptb- #2

500ng X (μL/123.1ng) = 4.06μL

Pptb- #3

500ng X (μL/92.7ng) = 5,39μL

Pptb- #4

500ng X (μL/74.5ng) = 6.71μL

Pptb- #5

500ng X (μL/114.6ng) = 4.36μL

Pptb- #7

500ng X (μL/154.8ng) = 3.23μL

Pptb- #14

500ng X (μL/127.9ng) = 3.91μL

Pptb- #15

500ng X (μL/110.4ng) = 4.53μL

Pptb- #16

500ng X (μL/112.4ng) = 4.45μL

Pptb- #17

500ng X (μL/124.5ng) = 4.01μL

Pptb- #18

500ng X (μL/219.8ng) = 2.27μL

Pptb- #21

500ng X (μL/157.6ng) = 3.17μL

Pptb- #22

500ng X (μL/145.6ng) = 3.43μL

Pptb- #23

500ng X (μL/134.2ng) = 3.73μL

Pptb- #24

500ng X (μL/114.6ng) = 4.36μL

Pptb- #25

500ng X (μL/189.8ng) = 2.63μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || Pptb- #1 | 2μL | 2μL | 2μL | 4.78μL | 9.22μL | 20μL || Pptb- #2 | 2μL | 2μL | 2μL | 4.06μL | 9.94μL | 20μL || Pptb- #3 | 2μL | 2μL | 2μL | 5.39μL | 8.61μL | 20μL || Pptb- #4 | 2μL | 2μL | 2μL | 6.71μL | 7.29μL | 20μL || Pptb- #5 | 2μL | 2μL | 2μL | 4.36μL | 9.64μL | 20μL || Pptb- #7 | 2μL | 2μL | 2μL | 3.23μL | 10.77μL | 20μL || Pptb- #14 | 2μL | 2μL | 2μL | 3.91μL | 10.09μL | 20μL || Pptb- #815 | 2μL | 2μL | 2μL | 4.53μL | 9.47μL | 20μL || Pptb- #16 | 2μL | 2μL | 2μL | 4.45μL | 9.55μL | 20μL || Pptb- #17 | 2μL | 2μL | 2μL | 4.02μL | 9.98μL | 20μL || Pptb- #18 | 2μL | 2μL | 2μL | 2.27μL | 11.73μL | 20μL || Pptb- #21 | 2μL | 2μL | 2μL | 3.17μL | 10.83μL | 20μL || Pptb- #22 | 2μL | 2μL | 2μL | 3.43μL | 10.57μL | 20μL || Pptb- #23 | 2μL | 2μL | 2μL | 3.73μL | 10.27μL | 20μL || Pptb- #24 | 2μL | 2μL | 2μL | 4.36μL | 9.64μL | 20μL || Pptb- #25 | 2μL | 2μL | 2μL | 2.63μL | 11.37μL | 20μL ||

Heat shock for 45 minutes in water bath and 15 minutes at 80°C on heat block to inactivate EcoR1

[Back to top](#top)

#

09/6/13

**Quantification and DNA Extraction of 3A**

QIAprep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/1/1d/86.png)

![](<p align=)
![HTML tutorial](https://static.igem.org/mediawiki/2013/0/06/87.png)

" alt="HTML tutorial" width="300" height="300" >

[Back to top](#top)

#

09/5/13

**Gel of Plep #1**

| Row 1 || Lane Number | Sample || 1 | Ladder (1kb plus) || 2 | Plep #1 || 3 | Empty || 4 | Empty || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/6/60/85.png)

[Back to top](#top)

#

09/5/13

**Digest of Plep #1**

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Amount | Component || 6.67μL | Plep || 2μL | 10x Buffer || 2μL | EcoR1 || 2μL | Pst1 || 7.33μL | PCR Water ||

500ng X (μL/75ng) = 6.67μL

[Back to top](#top)

#

09/5/13

**Quantification and DNA Extraction of 3A**

QIAprep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/d/dc/82.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/1/16/83.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/8/82/84.png)

[Back to top](#top)

#

09/4/13

**Gel of Colony PCR**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | T2 #1 || 3 | T2 #2 || 4 | T2 #3 || 5 | T2 #4 || 6 | Ter #5 || 7 | T2 #16 || 8 | T2 #17 || 9 | T2 #18 || 10 | T2 #19 || 11 | T2 #20 || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || Row #2 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Pveg2 #1 || 3 | Pveg2 #2 || 4 | Pveg2 #3 || 5 | Pveg2 #4 || 6 | Pveg2 #5 || 7 | Pveg2 #16 || 8 | Pveg2 #17 || 9 | Pveg2 #18 || 10 | Pveg2 #19 || 11 | Pveg2 #20 || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || | | | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/e/ec/81.png)

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Pptb- #1 || 3 | Pptb- #2 || 4 | Pptb- #3 || 5 | Pptb- #4 || 6 | Pptb- #5 || 7 | Pptb- #16 || 8 | Pptb- #17 || 9 | Pptb- #18 || 10 | Pptb- #19 || 11 | Pptb- #20 || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || Row #2 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Plep #1 || 3 | Plep #2 || 4 | Plep #3 || 5 | Plep #4 || 6 | Plep #5 || 7 | Plep #16 || 8 | Plep #17 || 9 | Plep #18 || 10 | Plep #19 || 11 | Plep #20 || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || | | | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/f/f7/80.png)

[Back to top](#top)

#

09/1/13

**3A Ligation**

1) Clos Ori +Ptb- + pSB1A3

1) Clos Ori +Pveg1 + pSB1A3

1) Clos Ori +Pveg2 + pSB1A3

1) Clos Ori +Pliag + pSB1A3

1) mlSR + Plep + pSB1A3

1) mlSR +Terminator2 (4P) + pSB1A3

#

08/30/13

**Gel of 3A Assembly**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | 2 #8 |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | 2 #10 || 3 | 2 #12 || 4 | 3 #15 || 5 | 3 #17 || 6 | 4 #1 || 7 | 4 #3 || 8 | 5 #1 || 9 | 6 #1 || 10 | 6 #3 || 11 | Ladder (1kb plus) || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/7/74/74.png)

[Back to top](#top)

#

08/30/13

**Digest of 3A Assembly**

3 #17

500ng X (μL/123.2ng) = 4.06μL

6 #1

500ng X (μL/98.7ng) = 5.07μL

6 #3

500ng X (μL/110.2ng) = 4.54μL

5 #1

500ng X (μL/162.6ng) = 3.08μL

2 #12

500ng X (μL/78.8ng) = 6.35μL

3 #15

500ng X (μL/131.8ng) = 3.79μL

4 #1

500ng X (μL/146.5ng) = 3.41μL

2 #8

500ng X (μL/117.7ng) = 4.25μL

2 #10

500ng X (μL/87.3ng) = 5.73μL

4 #3

500ng X (μL/82.9ng) = 6.03μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || 3 #17 | 2μL | 2μL | 2μL | 4.06μL | 9.94μL | 20μL || 6 #1 | 2μL | 2μL | 2μL | 5.07μL | 8.93μL | 20μL || 6 #3 | 2μL | 2μL | 2μL | 4.54μL | 9.46μL | 20μL || 5 #1 | 2μL | 2μL | 2μL | 3.08μL | 10.92μL | 20μL || 2 #12 | 2μL | 2μL | 2μL | 6.35μL | 7.65μL | 20μL || 3 #15 | 2μL | 2μL | 2μL | 3.79μL | 10.21μL | 20μL || 4 #1 | 2μL | 2μL | 2μL | 3.41μL | 10.59μL | 20μL || 2 #8 | 2μL | 2μL | 2μL | 4.25μL | 9.75μL | 20μL || 2 #10 | 2μL | 2μL | 2μL | 5.73μL | 8.27μL | 20μL || 4 #3 | 2μL | 2μL | 2μL | 6.03μL | 7.97μL | 20μL ||

Heat shock for 45 minutes in water bath and 15 minutes at 80°C on heat block to inactivate EcoR1

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#

08/30/13

**Quantification and DNA Extraction of 3A**

QIAprep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/5/55/68.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/1/1f/69.png)

[Back to top](#top)

#

08/27/13

**Gel of 3A Assembly**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder (1kb plus) |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | 1 #9 || 3 | 2 #22 || 4 | 2 #24 || 5 | 2 #26 || 6 | 3 #3 || 7 | 4 #2 || 8 | 5 #3 || 9 | 5 #11 || 10 | 6 #2 || 11 | 6 #19 || 12 | 6 #24 || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || | |

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#

08/26/13

**Digest of Ter and Adh6**

1 #9

500ng X (μL/124.7ng) = 4μL

ter #18

500ng X (μL/118.9ng) = 4.21μL

2 #22

500ng X (μL/76.1ng) = 6.57μL

2 #24

500ng X (μL/96.4ng) = 5.19μL

2 #26

500ng X (μL/99.4ng) = 5.03μL

3 #3

500ng X (μL/101ng) = 4.95μL

4 #2

500ng X (μL/104ng) = 4.81μL

5 #3

500ng X (μL/84.6ng) = 5.91μL

5 #11

500ng X (μL/124.6ng) = 4.01μL

6 #2

500ng X (μL/78.8ng) = 6.35μL

6 #19

500ng X (μL/141.4ng) = 3.54μL

6 #24

500ng X (μL/100.2ng) = 4.99μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || 1 #9 | 2μL | 2μL | 2μL | 4.21μL | 9.79μL | 20μL || 2 #22 | 2μL | 2μL | 2μL | 6.57μL | 7.43μL | 20μL || 2 #24 | 2μL | 2μL | 2μL | 5.19μL | 8.81μL | 20μL || 2 #26 | 2μL | 2μL | 2μL | 5.03μL | 8.97μL | 20μL || 3 #3 | 2μL | 2μL | 2μL | 4.95μL | 9.05μL | 20μL || 4 #2 | 2μL | 2μL | 2μL | 4.81μL | 9.19μL | 20μL || 5 #3 | 2μL | 2μL | 2μL | 5.91μL | 8.09μL | 20μL || 5 #11 | 2μL | 2μL | 2μL | 4.01μL | 9.99μL | 20μL || 6 #2 | 2μL | 2μL | 2μL | 6.35μL | 7.65μL | 20μL || 6 #19 | 2μL | 2μL | 2μL | 3.54μL | 10.46μL | 20μL || 6 #24 | 2μL | 2μL | 2μL | 4.99μL | 9.01μL | 20μL ||

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h1>

08/27/13

**Quantification of 3A Assembly**

QUIAPREP/Spin Miniprep Kit < (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/9/99/66.png)

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#

08/26/13

**Gel of ter and Adh6**

| Row 1 || Lane Number | Sample || 1 | Ladder (1kb plus) || 2 | ter #6 || 3 | ter #18 || 4 | ter #24 || 5 | Adh6 #8 || 6 | Adh6 #14 || 7 | Adh6 #15 || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

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#

08/26/13

**Digest of Ter and Adh6**

ter #6

500ng X (μL/124.7ng) = 4μL

ter #18

500ng X (μL/108ng) = 4.63μL

ter #24

500ng X (μL/315ng) = 1.59μL

Adh6 #8

500ng X (μL/300.3ng) = 12.41μL

Adh6 #14

500ng X (μL/95.4ng) = 5.24μL

Adh6 #15

500ng X (μL/328.3ng) = 1.52μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || ter #6 | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL || ter #18 | 2μL | 2μL | 2μL | 4.63μL | 9.37μL | 20μL || ter #24 | 2μL | 2μL | 2μL | 1.59μL | 12.41μL | 20μL || Adh6 #8 | 2μL | 2μL | 2μL | 1.67μL | 12.33μL | 20μL || Adh6 #14 | 2μL | 2μL | 2μL | 5.24μL | 11.76μL | 20μL || Adh6 #15 | 2μL | 2μL | 2μL | 1.52μL | 12.48μL | 20μL |

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#

08/26/13

**DNA Extraction of Adh6 and Ter**

QIAprep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

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#

08/22/13

**Transformation of 3A Assembly**

1. Transform 4μL of ligation product into 100μL aliquots of competent E. coli cells
2. Incubate on ice for 30 minutes
3. Heat shock in water bath for 60 seconds
4. Incubate on ice for 5 minutes
5. Add 600μL of psi broth
6. Incubate at 37°C for 2 hours at 200 rpm
7. Make necessary dilutions and plate out

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#

08/22/13

**3A Ligation**

1) Clos Ori +Pveg2 (20G) + pSB1A3

1) Clos Ori +Plep (20E) + pSB1A3

1) Clos Ori +Pveg1 (23B) + pSB1A3

1) Clos Ori +Pliag (20A) + pSB1A3

1) mlSR +Terminator1 (6D) + pSB1A3

1) mlSR +Terminator2 (4P) + pSB1A3

Amount | Sample || 2μL | Upstream Part || 2μL | Downstream Part || 2μL | Destination Backbone || 2μL | 10x T4 DNA Ligase Buffer || 0.5μL | T4 DNA Ligase || 10μL | PCR Water ||

Incubate at room temperature for 10 minutes, then at 80°C for 20 minutes to inactivate EcoR1

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#

08/15/13

Clos Ori

500ng X (440bp/2000bp) X (μL/190.5ng) = 0.577μL 1:105.7μL

mlSR

500ng X (750bp/2000bp) X (μL/260.4ng) = 0.908μL 1:109.08μL

Pliag

500ng X (121bp/2000bp) X (μL/150.1ng) = 0.201μL 1:102.01μL

Pveg1

500ng X (100bp/2000bp) X (μL/90.0ng) = 0.278μL 1:102.78μL

Plep

500ng X (157bp/2000bp) X (μL/235.0ng) = 0.167μL 1:101.67μL

Pveg2

500ng X (237bp/2000bp) X (μL/192.3ng) = 0.308μL 1:103.08μL

Terminator

500ng X (50bp/2000bp) X (μL/108.6ng) = 0.115μL 1:101.15μL

Terminator

500ng X (50bp/2000bp) X (μL/131.8ng) = 0.095μL 1:100.95μL

pSB1A3

500ng X (μL/31.8ng) = 2μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | Xba1 | Spe1 | 100x Bovine Serum Albumin | 10x Buffer | DNA | PCR dH2O | Total || Clos Ori | 1μL | 1μL | - | - | 1μL | 5μL | 5.7μL | 36.3μL | 50μL || mlSR | 1μL | 1μL | - | - | 1μL | 5μL | 9.08μL | 32.92μL | 50μL || Pliag | - | - | 1μL | 1μL | 1μL | 5μL | 5.7μL | 39.99μL | 50μL || Pveg1 | - | - | 1μL | 1μL | 1μL | 5μL | 2.78μL | 39.22μL | 50μL || Pveg2 | - | - | 1μL | 1μL | 1μL | 5μL | 1.67μL | 40.33μL | 50μL || Plep | - | - | 1μL | 1μL | 1μL | 5μL | 3.08μL | 38.92μL | 50μL || Terminator 6D | - | - | 1μL | 1μL | 1μL | 5μL | 1.15μL | 40.85μL | 50μL || Temrinator 4P | - | - | 1μL | 1μL | 1μL | 5μL | 0.95μL | 41.05μL | 50μL || pSB1A3 | 1μL | 1μL | - | - | 1μL | 5μL | 16μL | 26μL | 50μL ||

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#

08/21/13

**PCR of BktB Fragments 1 & 2 used for SDM Step1**

Previous attempt produced a lot of extraneous artifacts.

Primer concentration was too high and so was the template concentrations

Used Grad program previously used to run PCR machine

50°C to 60°V used 4 and 0

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#

08/21/13

**Quantification of Backbones Post-SPRI Based Size Selection**

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

The concentrations of pSB1A3 is low. Therefore, the SPRI based size selection was conducted on it once more. The gel results are displayed below.

![HTML tutorial](https://static.igem.org/mediawiki/2013/3/38/60.1.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/e/e6/60.png)

[Back to top](#top)

#

08/21/13

**Transformation of Ter + pSB1C3 and Adh6 + pSB1C3**

1. Thaw competent Top10 cell of E. coli from -80°C (100μL aliquots)
2. Add 20μL of ligation product to Top 10 E. coli
3. Incubate on ice for 30 minutes
4. Heat shock in water bath for exactly 60 seconds
5. Incubate on ice for 5 minutes
6. Add 600μL of psi broth
7. Incubate at 37°C for 2 hours at 200 rpm
8. Make dilutions using 90μL of psi broth: 1:10, 1:100, 1:1000
9. Plate out 25μL undiluted and diluted solutions onto a petri dish

| Row 1 || Lane Number | Sample || 1 | Ladder (1kb plus) || 2 | pSB1A3 || 3 | Empty || 4 | Empty || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

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#

08/21/13

**Gel Post SPRI Based Size Selection**

| Row 1 || Lane Number | Sample || 1 | Ladder (1kb plus) || 2 | pSB1A3 || 3 | pSB1C3 || 4 | pSB1K3 || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

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#

08/21/13

**SPRI Based Size Selection**

Use the instructions labeled “left side size slection.” This kit will hopefully remove the primer dimer from our product. Remove the primer dimer from the backbone PCR clean-up.

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#

08/21/13

**Gel of Refined Backbone PCR**

| Row 1 || Lane Number | Sample || 1 | Ladder (1kb plus) || 2 | pSB1A3 || 3 | pSB1C3 || 4 | pSB1K3 || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

[Back to top](#top)

#

08/21/13

**PCR of pSB1A3, pSB1K3, pSB1K3 (refined)**

Sample G in the primer matrix will be used as a template for this particular PCR

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Label | Amount | Component || PCR # 1 | 3μL | SBprep-3P-1 ||  | 1μL | SBprep-2Ea ||  | 2.0μL | pSB1A3 ||  | 94μL | PCR Supermix High Fidelity || PCR #3 | 3μL | SBprep-3P-1 ||  | 1μL | SBprep-2Ea ||  | 2.0μL | pSB1C3 || PCR #2 | 3μL | SBprep-3P-1 ||  | 1μL | SBprep-2Ea ||  | 2.0μL | pSB1K3 ||  | 94μL | PCR Supermix High Fidelity |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

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#

08/20/13

**Gel of Fifth PCR of Backbones**

| Row 1 || Lane Number (1kb plus) | Sample || 1 | Ladder || 2 | E || 3 | G || 4 | Empty || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c2/41.png)

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#

08/15/13

Use the two concentrations of E and G on 20-August

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/17.png)

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#

08/20/13

**PCR of Backbone Discussion**

After discussing the results with Dr. Wawrik, it has been decided to cut the number of cycles down to thirty, and to increase the template used by two.

The best concentrations of primers to be used are indicated by the results of the primer matrix on letters E and G. F appeared to have been a probable pipetting error.

List of reactants and amount for primer matrix PCR optimization

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Label | Amount | Component || E | 0.5μL | SBprep-3P-1 (1:2 dilutions) use 1μL ||  | 1.5μL | SBprep-2Ea || 1.0μL | Template ||  | 46.5μL | PCR Supermix High Fidelity || G | 1.5μL | SBprep-3P-1 ||  | 0.5μL | SBprep-2Ea (1:2 dilutions) use 1μL ||  | 1.0μL | Template ||  | 46.5μL | PCR Supermix High Fidelity |

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#

08/19/13

**Gel of Ter and Adh6 Post Digest**

| Row 1 || Lane Number (1kb plus) | Sample || 1 | Ladder || 2 | Adh6 #19 || 3 | Adh6 #3 || 4 | Ter #5 || 5 | Ter #23 || 6 | Ter #26 || 7 | Ter #9 || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c2/41.png)

[Back to top](#top)

#

08/14/13

**Gel of pSB1K3, pSB1C3, pSB1A3 Stocks from 9-May**

| Row 1 || Lane Number (1kb plus) | Sample || 1 | Ladder || 2 | pSB1A3 || 3 | pSB1C3 || 4 | pSB1K3 || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/4/4d/40.png)

[Back to top](#top)

#

08/14/13

**PCR Clean-up Quanitification**

![HTML tutorial](https://static.igem.org/mediawiki/2013/4/49/39.1.png)

Although the concentrations are good, the purity is still in question. It is an improvement on the PCR from 13-August

One possibility is that the original stocks used as templates in the PCR may not be pure enough. Therefore, a gel will be run on these original stocks

[Back to top](#top)

#

08/14/13

**Gel of pSB1K3, pSB1C3, pSB1A3 Post Clean-up**

| Row 1 || Lane Number (1kb plus) | Sample || 1 | Ladder || 2 | pSB1A3 || 3 | pSB1C3 || 4 | pSB1K3 || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/6/68/39.png)

[Back to top](#top)

#

08/14/13

**PCR Clean-up of the Three Backbones**

QIAquick PCR purification kit (250) is used before viewing the post clean-up product on a gel

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#

08/14/13

**Gel of BktB Fragments**

| Row 1 || Lane Number (1kb plus) | Sample || 1 | Ladder || 2 | 50°C || 3 |  || 4 |  || 5 | 60°C || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

What happened here? What is in the lanes above the comb?

Many have some product and we will use the forward and reverse primers to get the whole gene, if present

![HTML tutorial](https://static.igem.org/mediawiki/2013/8/87/38.png)

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#

08/14/13

**Gel of pSB1K3, pSB1C3, pSB1A3 Pre Clean-up**

| Row 1 || Lane Number (1kb plus) | Sample || 1 | Ladder || 2 | pSB1A3 || 3 | pSB1C3 || 4 | pSB1K3 || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/3/3f/37.1.png)

[Back to top](#top)

#

08/14/13

**Quantified Backbone Stocks for PCR**

The following stock are to be used for backbone amplification in the PCR procedure illustrated on 14-August

![HTML tutorial](https://static.igem.org/mediawiki/2013/8/87/37.png)

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#

08/14/13

**Preparing an Equimolar solution of BktB Fragments for PCR to Stitch**

Fragment 1: 340bp – 125.6ng/μL

Fragment 1: 660bp – 66.3ng/μL

Fragment 1: 300bp – 231.7ng/μL

Use 1μL of fragment 3 = 231.7ng/μL

Fragment 2:

(221.7) X (660/300) = 520ng x (1μL/66.3ng) = 7.7μL

Fragment 3:

(221.7) X (340/300) = 263ng x (1μL/125.6ng) = 2.1μL

Added to a 1.5mL epi tube and diluted to 200μL

1004ng of DNA/ 200μL = 5ng/μL final concentration of DNA
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#

08/14/13

**PCR of i-GEM Plasmid Backbones Continued**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PCR #1 || Amount (μL) | Component || 100 | PCR Supermix High Fidelity || 0.7 | SB-prep-3P-1 || 0.7 | SB-prep-2Ea || 0.6 | pSB1A3 (1ng/μL) || PCR #2 || Amount (μL) | Component || 100 | PCR Supermix High Fidelity || 0.7 | SB-prep-3P-1 || 0.7 | SB-prep-2Ea || 0.6 | pSB1C3 (1ng/μL) || PCR #3 || Amount (μL) | Component || 100 | PCR Supermix High Fidelity || 0.7 | SB-prep-3P-1 || 0.7 | SB-prep-2Ea || 0.6 | pSB1K3 (1ng/μL) ||

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c1/35.png)

[Back to top](#top)

#

08/14/13

**Adjusted PCR of iGEM Plasmid Backbones**

Diluting concentrations of pSB1A3, pSB1C3, pSB1K3 to 1ng/μL

Quantification of current backbones to confirm concentrations before conducting dilution

![HTML tutorial](https://static.igem.org/mediawiki/2013/0/0a/34.png)

pSB1A3

Vf = (27.5ng/μL) X (1μL) X (1μL/1ng) = 27.5μL – 1μL of template = 26.5μL of PCR water

pSB1C3

Vf = (39.9ng/μL) X (1μL) X (1μL/1ng) = 39.9μL – 1μL of template = 38.9μL of PCR water

pSB1K3

Vf = (29.4ng/μL) X (1μL) X (1μL/1ng) = 29.4μL – 1μL of template = 28.4μL of PCR water

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#

08/13/13

**Quantification of PCR Clean-up**

The concentrations are good, however the purity of the product is not. Actions taken to remedy this problem are to run another PCR using 1ng/μL of template and running the third step of the PCR cycle at 68°C for 5 minutes.

![HTML tutorial](https://static.igem.org/mediawiki/2013/9/9c/33.png)

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#

08/13/13

**Gel of pSB1K3, pSB1C3, pSB1A3 Post Clean-up**

| Row 1 || Lane Number | Sample || 1 | Ladder || 2 | pSB1A3 || 3 | pSB1C3 || 4 | pSB1K3 || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/ce/32.1.png)

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#

08/13/13

**PCR Clean-up of pSB1K3, pSB1C3, pSB1A3**

The three backbones were cleaned up using QIAquick PCR Purification Kit (250) so that the next gel will much more clearly display the backbone.

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#

08/13/13

**Gel of pSB1K3, pSB1C3, pSB1A3**

| Row 1 || Lane Number | Sample || 1 | Ladder || 2 | pSB1K3 || 3 | pSB1C3 || 4 | pSB1A3 || 5 | Empty || 6 | Empty || 7 | Empty || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial](https://static.igem.org/mediawiki/2013/1/16/32.png)

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h1>

08/13/13

**Plasmid Extraction of AaDc-, AaDc+, Thl+, Thl-, and Ptb+**

QUIAPREP/Spin Miniprep Kit < (250)

Cells were centrifuged from an inoculated broth

![HTML tutorial](https://static.igem.org/mediawiki/2013/1/19/30.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/6/68/30.1.png)

None of the concentrations obtained are high enough to work with. Must go about finding a more efficient plasmid extraction for these samples.

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#

08/13/13

**PCR of i-GEM Plasmid Backbones**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PCR #1 || Amount (μL) | Component || 100 | PCR Supermix High Fidelity || 0.7 | SB-prep-3P-1 || 0.7 | SB-prep-2Ea || 0.6 | pSB1C3 (43ng/μL) || PCR #2 || Amount (μL) | Component || 100 | PCR Supermix High Fidelity || 0.7 | SB-prep-3P-1 || 0.7 | SB-prep-2Ea || 0.6 | pSB1C3 (74.8ng/μL) || PCR #3 || Amount (μL) | Component || 100 | PCR Supermix High Fidelity || 0.7 | SB-prep-3P-1 || 0.7 | SB-prep-2Ea || 0.6 | pSB1C3 (55.1ng/μL) ||

![HTML tutorial](https://static.igem.org/mediawiki/2013/5/50/29.png)

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#

08/14/13

**Post-Cleanup of BktB PCR Products**

All Fragment 1’s, lanes 8 and 9 of Fragment 2, and all Fragment

![HTML tutorial](https://static.igem.org/mediawiki/2013/2/22/28.1.png)

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#

08/13/13

**PCR of BktB from PCR Tempalte**

![HTML tutorial](https://static.igem.org/mediawiki/2013/b/b9/28.png)

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#

08/9/13

**Gel of BktB Fragments**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample | Temperature (°C) || 1 | Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Fragment 1 | 50 || 3 | Fragment 1 || 4 | Fragment 1 || 5 | Fragment 1 | 60 || 6 | Fragment 2 | 50 || 7 | Fragment 2 || 8 | Fragment 2 || 9 | Fragment 2 | 60 || 10 | Fragment 3 | 50 || 11 | Fragment 3 || 12 | Fragment 3 || 13 | Fragment 3 | 60 || 14 | Empty || 15 | EMpty || 16 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/f/ff/27.png)

Based on 2% agarose gel, 115V for about 30 minutes

Going to PCR the whole gene to see if I can reduce non-specific products in fragment #2

![HTML tutorial](https://static.igem.org/mediawiki/2013/7/79/27.1.png)

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#

08/8/13

**PCR of BktB Fragments**

Preformed gradient PCR for all 3 BktB fragments

Undiluted template was used

![HTML tutorial](https://static.igem.org/mediawiki/2013/2/25/26.png)

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#

08/7/13

**Transformation of Top10 with ter in pSB1C3**

1. Thaw competent 100μL aliquot of E. coli cells from -80°C freezer
2. Add 20μL of ligation poduct to Top10 E. coli
3. Incubate on ice for 30 minutes
4. Heat shock in water bath for 60 seconds
5. Incubate on ice for 5 minutes
6. Add 600μL of psi broth
7. Incubate at 37°C for 2 hours at 200 rpm
8. Make a dilution series of 1:10, 1:100, 1:1000, and 1:10000

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#

08/7/13

**Ligation of pSB1C3 and Adh6**

Into 1.5mL microcentrifuge tubes

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Amount (μL) | Component || 20 | Heat killed digest of pSB1C3 || 20 | Heat killed digest of Adh6 || 1.0 | T4 Ligase || 5.0 | 10X T4 Ligase Buffer || 4.0 | Nuclease Free PCR Water || 50 | Total ||

Incubate on bench for 45 minutes

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#

08/9/13

**digestion of ter**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || ter #23 | 1μL | 1μL | 1μL | 2.5μL | 14.5μL | 20μL || ter #7 | 1μL | 1μL | 1μL | 1.5μL | 15.5μL | 20μL || ter #26 | 1μL | 1μL | 1μL | 2μL | 15μL | 20μL || ter #5 | 1μL | 1μL | 1μL | 2μL | 15μL | 20μL || pSB1C3 | 1μL | 1μL | 1μL | 2.5μL | 14.5μL | 20μL ||

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#

08/9/13

**Plasmid Extraction of AaDc- and Ptb-**

![HTML tutorial](https://static.igem.org/mediawiki/2013/7/71/21.png)

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#

08/7/13

**Restriction Digest of Ccell Adh1 and Adh2**

Adh1 #5

500ng X (μL/297.2ng) = 1.68μL

Adh1 #19

500ng X (μL/89.3ng) = 5.60μL

Adh1 #24

500ng X (μL/261.6ng) = 1.91μL

Adh1 #4

500ng X (μL/264.0ng) = 1.89μL

Adh1 #13

500ng X (μL/588.6ng) = 0.85μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || Adh1 #5 | 2μL | 2μL | 2μL | 1.5μL | 12.5μL | 20μL || Adh1 #19 | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL || Adh1 #24 | 2μL | 2μL | 2μL | 2μL | 12μL | 20μL || Adh1 #4 | 2μL | 2μL | 2μL | 2μL | 12μL | 20μL || Adh1 #13 | 2μL | 2μL | 2μL | 1μL | 13μL | 20μL ||

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || Adh6 #5 | 1μL | 1μL | 1μL | 5.0μL | 12.0μL | 20μL || Adh6 #19 | 1μL | 1μL | 1μL | 2.0μL | 15.0μL | 20μL || Adh6 #3 | 1μL | 1μL | 1μL | 3.0μL | 14.0μL | 20μL || Adh6 #15 | 1μL | 1μL | 1μL | 3.0μL | 14.0μL | 20μL || pSB1C3 | 1μL | 1μL | 1μL | 2.5μL | 14.5μL | 20μL ||

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#

08/7/13

**Gel for Post-Digest of Promoters and Terminators**

Using 2% agarose in the gel

| Gel 1 || Lane Number | Sample || 1 | Ladder || 2 | #16-6D || 3 | #16-23B || 4 | #9-4P || 5 | #9-6D || 6 | #3-23B || 7 | #14-20E || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial]( https://static.igem.org/mediawiki/2013/8/88/18.png
)

| Gel 2 || Lane Number | Sample || 1 | Ladder || 2 | #15-20A || 3 | #7-20G || 4 | #7-4D || 5 | #14-20A || 6 | #10-4P || 7 | #11-20E || 8 | Empty ||

| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |

![HTML tutorial]( https://static.igem.org/mediawiki/2013/4/40/18.1.png
)

Samples display a possible insert, and will be sent off for sequencing

3-23B, 16-23B, 14-20E, 15-20A, 16-6D, 14-20A, and 10-4P

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#

08/7/13

**Modified Restriction Digest of Promoters and Terminators**

![HTML tutorial]( https://static.igem.org/mediawiki/2013/c/c9/17.png
)

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || #16-6D | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #16-23B | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #9-4P | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #9-6D | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #3-23B | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #14-20E | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #15-20A | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #7-20G | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #7-4D | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #14-20A | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #10-4P | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL || #11-20E | 1μL | 1μL | 1μL | 10.0μL | 7.0μL | 20μL ||

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#

08/7/13

**Plasmid Extraction of Ccell of Adh1 and Adh2**

QIAPrep Spin Miniprep Kit (250)

The gene’s origin is unknown, and will be found upon sequencing

![HTML tutorial]( https://static.igem.org/mediawiki/2013/c/c9/17.png
)

[Back to top](#top)

#

07/18/13

**Plasmid Prep of Transformed Top10 Cells with pSB1C3 + mlsR and puc19 + KIVD**

[Back to top](#top)

#

07/17/13

**DNA Extraction from Ca, Cs, Cb, and Cc using MoBio Power Soil DNA Extraction Kit**

[Back to top](#top)

#

07/11/13

**Dual Digest of pSB1C3 and mlsR Part**

[Back to top](#top)

#

07/25/13

**Gel of Aadc-/+, thl+/-, Ptb-/+ from 1 Aug**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Aadc- #6 || 3 | Aadc- #23 || 4 | Aadc+ #20 || 5 | thl- #4 || 6 | Aadc+ #15 || 7 | Aadc+ #2 || 8 | thl+ #17 || 9 | thl+ #3 || 10 | thl+ #10 || 11 | thl- #14 || 12 | thl- #21 || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || Row #2 || Lane Number | Sample || 1 | Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Empty || 3 | Empty || 4 | Empty || 5 | Empty || 6 | Empty || 7 | Ladder || 8 | Ptb- #5 || 9 | Ptb+ #9 || 10 | Ptb+ #1 || 11 | Ptb+ #26 || 12 | Ptb- #18 || 13 | Ptb- #8 || 14 | Empty || 15 | Empty || 16 | Empty || | | | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/d/db/146.png)

[Back to top](#top)

#

08/02/13

**Plasmid Extraction of Ccell Adh and Csac Adh**

QIAPrep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c7/145.png)

[Back to top](#top)

#

08/02/13

**Restriction Digest of Aadc-/+ and Ptb-/+**

#2 Aadc+ 500ng (1&#956L⁄85.9ng) = 6.0μL

#15 Aadc+ 500ng (1&#956L⁄117.5ng) = 4.5μL

#20 Aadc+ 500ng (1&#956L⁄92.1ng) = 5.5μL

#23 Aadc- 500ng (1&#956L⁄184.8ng) = 3.0μL

#6 Aadc- 500ng (1&#956L⁄218.1ng) = 2.5μL

#17 thl+ 500ng (1&#956L⁄100.0ng) = 5.0μL

#3 thl+ 500ng (1&#956L⁄84.4ng) = 6.0μL

#10 thl+ 500ng (1&#956L⁄90.4ng) = 5.5μL

#14 thl- 500ng (1&#956L⁄85.7ng) = 6.0μL

#21 thl- 500ng (1&#956L⁄89.1ng) = 5.5μL

#4 thl- 500ng (1&#956L⁄131.8ng) = 4.0μL

#9 Ptb+ 500ng (1&#956L⁄81.4ng) = 6.0μL

#1 Ptb+ 500ng (1&#956L⁄124.2ng) = 4.0μL

#26 Ptb+ 500ng (1&#956L⁄206.7ng) = 2.5μL

#18 Ptb- 500ng (1&#956L⁄103.7ng) = 5.0μL

#8 Ptb- 500ng (1&#956L⁄150.1ng) = 3.5μL

#5 Ptb- 500ng (1&#956L⁄216.0ng) = 2.5μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || #2 Aadc+ | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL || #15 Aadc+ | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL || #20 Aadc+ | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL || #23 Aadc- | 2μL | 2μL | 2μL | 3.0μL | 11.0μL | 20μL || #6 Aadc- | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL || #17 thl+ | 2μL | 2μL | 2μL | 5.0μL | 9.0μL | 20μL || #3 thl+ | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL || #10 thl+ | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL || #14 thl- | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL || #21 thl- | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL || #4 thl- | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL || #9 Ptb+ | 2μL | 2μL | 2μL | 6.0μL | 8.0μL | 20μL || #1 Ptb+ | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL || #26 Ptb+ | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL || #18 Ptb- | 2μL | 2μL | 2μL | 5.0μL | 9.0μL | 20μL || #8 Ptb- | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL || #5 Ptb- | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL ||

[Back to top](#top)

#

08/02/13

**Plasmid Extraction of Aadc-/+, Ptb-/+, thl-/+**

QIAPrep Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/0/07/140.1.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/6/67/141.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/d/d3/142.png)

[Back to top](#top)

#

08/01/13

**Gel of Regulatory Promoters and Terminators (1 Aug)**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1 || Lane Number | Sample || 1 | Ladder || 2 | 3:23B || 3 | 10:4P || 4 | 12:20G || 5 | 9:6D || 6 | 16:6D || 7 | 8:20G || 8 | 11:20E || 9 | 14:20E || 10 | 15:20A || 11 | 14:20A || 12 | 9:4P || 13 | 16:23B || 14 | 9:20G || 15 | 7:20G |

![HTML tutorial](https://static.igem.org/mediawiki/2013/0/09/140.png)

[Back to top](#top)

#

08/1/13

**Restriction Digest using EcoR1 and Pst1 (26 July Quantification)**

#16-6D 500ng (1&#956L⁄108.6ng) = 4.5μL

#14-20E 500ng (1&#956L⁄235.2ng) = 2.0μL

#12-20G 500ng (1&#956L⁄135.1ng) = 3.5μL

#8-20G 500ng (1&#956L⁄192.3ng) = 2.5μL

#14-20A 500ng (1&#956L⁄156.5ng) = 3.0μL

#9-6D 500ng (1&#956L⁄`99.7ng) = 2.5μL

#10-4P 500ng (1&#956L⁄131.8ng) = 4.0μL

#7-20G 500ng (1&#956L⁄232.8ng) = 2.0μL

#3-23B 500ng (1&#956L⁄90.0ng) = 5.5μL

#16-23B 500ng (1&#956L⁄123.5ng) = 4.0μL

#9-4P 500ng (1&#956L⁄145.2ng) = 3.5μL

#9-20G 500ng (1&#956L⁄106.8ng) = 4.5μL

#15-20A 500ng (1&#956L⁄150.1ng) = 3.5μL

#11-20E 500ng (1&#956L⁄126.2ng) = 4.0μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || #16-6D | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL || #14-20E | 2μL | 2μL | 2μL | 2.0μL | 12.0μL | 20μL || #12-20G | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL || #8-20G | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL || #14-20A | 2μL | 2μL | 2μL | 3.0μL | 11.0μL | 20μL || #9-6D | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL || #10-4P | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL || #7-20G | 2μL | 2μL | 2μL | 2.0μL | 12.0μL | 20μL || #3-23B | 2μL | 2μL | 2μL | 5.5μL | 8.5μL | 20μL || #16-23B | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL || #9-4P | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL || #9-20G | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL || #15-20A | 2μL | 2μL | 2μL | 3.5μL | 10.5μL | 20μL || #11-20E | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL ||

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#

08/1/13

**Plasmid Extraction of Terminators and Promoters**

QIAPrep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/d/de/136.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/9/9d/137.png)

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#

08/01/13

**Ampicillin 1x Preparation**

To make 40mL of ampicillin 1x antibiotic

1. 2.0g ampicillin 1000x stock from 4°C refrigerator
2. Pour the amipicillin ino a 50mL falcon
3. Fill flacon wih dH2O to 40mL mark
4. Use 10mL syringe with puradisc 25mm filter to dispense 10mL of ampicillin 1x into four 10mL falcons (leaves alternative stocks if one is contaminated)

Ampicillin 1000x = 50ng/mL

50ng/mL x 40mL = 2.0g Ampicillin 1000x

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#

07/31/13

**Promoter and Terminator Culture Preparation**

Used 2XYT liquid medium instead of LB to increase carbon amount in order to enhance growth of cultures.

-3mL of 2XYT with Chloramphenicol antibiotic

-Pellet of culture

Incubated at 37°C with shaking at 220rpm

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#

07/31/13

**2XYT Medium Preparation**

* (Agar: 10g)* Bacto Tryptone: 16g
  * Yeast Extract: 10g
  * Sodium Chloride: 5g
  * Distilled Water: 1000mL

Fill 1L jar with 500mL of dH2O. Pour components and magnetic stirring rod into jar. Fill jar with another 500mL of dH2O until the shoulder of the jar is reached.

Autoclave for 20 minutes at slow exhaust (Setting #2)

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#

07/31/13

**Gel of DNA Preps from R. eutropha**

Run a gel with 3μL of dye and 5μL of DNA extract for each of our R. eutropha extracts.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Phenol Chloroform (27 July) || 3 | R. eutropha 1 (24 July) || 4 | R. eutropha 2 (24 July) || 5 | Power Soil (25 July) || 6 | Phenol Chloroform (25 July) || 7 | Empty || 8 | Empty || | |

NOTE: Lane six shows chewed up DNA by nucleases

Disposed all samples except for Power Soil because it has the highest efficacy

![HTML tutorial](https://static.igem.org/mediawiki/2013/3/3e/134.png)

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#

07/30/13

**Plasmid Purification**

Plasmid purification of top 10 cells transformed with ligation of pSB1C3 and Ca promoters (Ptb-RBS, Ptb+RBS, thl+RBS, thl-RBS, Aadc+RBS, AaDc-RBS)

![HTML tutorial](https://static.igem.org/mediawiki/2013/6/60/132.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/6/6c/133.png)

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#

07/30/13

**Plasmid Extraction of Terminators and Promoters**

Used QIAPrep Spin Miniprep Kit (250)

![HTML tutorial](https://static.igem.org/mediawiki/2013/8/83/131.png)

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#

07/27/13

**Dual Re gel of Regulatory Terminators and Promoters**

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1|  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Lane Number | Sample || 1 | Ladder || 2 | #14-20A || 3 | #17-20G || 4 | #14-20E || 5 | #11-20E || |

![HTML tutorial](https://static.igem.org/mediawiki/2013/5/51/130.1.png)

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#

07/27/13

**Dual Digest of pSB1C3 and mlsR Part**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Lane Number | Sample || 1 | Ladder || 2 | #10-4p || 3 | #14-20E || 4 | #14-20A || 5 | #7-20G || 6 | #11-20E || 7 | #16-20E || 8 | #9-4P || 9 | #15-20A || |

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/ca/130.png)

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#

07/11/13

**Dual Digest of pSB1C3 and mlsR Part**

500ng in 20μL with 2μL for each digest

#11-20E 500ng (1&#956L⁄58.1ng) = 8.5μL

#9-4P 500ng (1&#956L⁄40.2ng) = 12.5μL

#14-20A 500ng (1&#956L⁄43.5ng) = 12.5μL

#7-20G 500ng (1&#956L⁄59.4ng) = 12.5μL

#14-20E 500ng (1&#956L⁄37.5ng) = 12.5μL

#10-4P 500ng (1&#956L⁄43.6ng) = 12.5μL

#15-20A 500ng (1&#956L⁄51.6ng) = 12.5μL

#16-23B 500ng (1&#956L⁄52.5ng) = 12.5μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || #11-20E | 2μL | 2μL | 2μL | 8.5μL | 5.5μL | 20μL || #9-4P | 2μL | 2μL | 2μL | 12.5μL | 1.5μL | 20μL || #14-20A | 2μL | 2μL | 2μL | 11.5μL | 2.5μL | 20μL || #7-20G | 2μL | 2μL | 2μL | 8.5μL | 5.5μL | 20μL || #14-20E | 2μL | 2μL | 2μL | 13.5μL | 0.5μL | 20μL || #10-4P | 2μL | 2μL | 2μL | 11.5μL | 2.5μL | 20μL || #15-20A | 2μL | 2μL | 2μL | 9.5μL | 4.5μL | 20μL || #16-23B | 2μL | 2μL | 2μL | 9.5μL | 4.5μL | 20μL ||

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#

07/26/13

**Nano Drop of Isolated DNA from July 25**

![HTML tutorial](https://static.igem.org/mediawiki/2013/a/af/126.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/b/be/127.png)

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#

07/26/13

**Gel of BktB piece 3 PCR using DNA Extracts**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample || 1 | Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | R. eutropha extract (27 July) || 3 | R. eutropha 1 (24 July) || 4 | R. eutropha 2 (24 July) || 5 | Power Soil (25 July) || 6 | Phenol Chloroform (25 July) || 7 | Empty || 8 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/2/28/125.png)

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#

07/26/13

**Gel Repeat of BktB PCR Gradient (25 July)**

Repeated gel with larger loading of DNA. Removing only loaded 5μL of dye pls products. Wanted to see if bands appear brighter.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample | Temperature (°c) || 1 | Kb Plus Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Piece 1 | 50 || 3 | Piece 1 | 52.7 || 4 | Piece 1 | 54.5 || 5 | Piece 1 | 56.4 || 6 | Piece 1 | 60 || 7 | Piece 2 | 50 || 8 | Piece 2 | 52.7 || 9 | Piece 2 | 54.5 || 10 | Piece 2 | 56.4 || 11 | Piece 2 | 60 || 12 | Ladder || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/8/81/123.png)

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample | Temperature (°c) || 1 | Kb Plus Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Piece 3 | 50 || 3 | Piece 3 | 52.7 || 4 | Piece 3 | 54.5 || 5 | Piece 3 | 56.4 || 6 | Piece 3 | 60 || 7 | Whole gene | 50 || 8 | Whole gene | 52.7 || 9 | Whole gene | 54.5 || 10 | Whole gene | 56.4 || 11 | Whole gene | 60 || 12 | Ladder || 13 | Empty || 14 | Empty || 15 | Empty || 16 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/a/a8/124.png)

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#

07/11/13

**PCR of BktB piece 3 using Different DNA Extraction**

Wanted to identify which extracts may have DNA present

Previous gradient seems to have produced a PCR product for 3 piece. Used temperature 55°C, since this is the predicted optimal temperature for the SDM2 forward and the BktB\_Rev primers

Templates used:

* R. eutropha = 27 July\_ 16.9
* R. eutropha = 24 July\_ 25
* R. eutropha = 24 July\_ 38.9
* Power Soil Extraction = 25 July\_ 28.3
* Phenol Chloroform Extraction = 25 July\_ 637

![HTML tutorial](https://static.igem.org/mediawiki/2013/b/be/122.png)

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#

07/25/13

**Gel of Gradient PCR for BktB Site-Directed Mutagenesis**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample | Temperature (°c) || 1 | Kb Plus Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Forward + SDM1 Reverse | 50 || 3 | Forward + SDM1 Reverse | 52.7 || 4 | Forward + SDM1 Reverse | 54.5 || 5 | Forward + SDM1 Reverse | 56.4 || 6 | Forward + SDM1 Reverse | 60 || 7 | SDM1 Forward + SDM2 Reverse | 50 || 8 | SDM1 Forward + SDM2 Forward | 52.7 || 9 | SDM1 Forward + SDM2 Reverse | 54.5 || 10 | SDM1 Forward + SDM2 Reverse | 56.4 || 11 | SDM2 Forward + Reverse | 60 || 12 | Empty || 13 | Empty || 14 | Empty || 15 | EMpty || 16 | Empty || Row #2 || Lane Number | Sample | Temperature (°C) || 1 | Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | SDM2 Forward + Reverse | 50 || 3 | SDM2 Forward + Reverse | 52.7 || 4 | SDM2 Forward + Reverse | 54.5 || 5 | SDM2 Forward + Reverse | 56.4 || 6 | SDM2 Forward + Reverse | 60 || 7 | Forward + Reverse | 50 || 8 | Forward + Forward | 52.7 || 9 | Forward + Reverse | 54.5 || 10 | Forward + Reverse | 56.4 || 11 | Forward + Reverse | 60 || 12 | Empty || 13 | Empty || 14 | Empty || 15 | Empty || 16 | Empty || | | | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/d/d2/121.png)

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#

07/25/13

**DNA Extraction from R. eutropha**

If DNA template is the problem with the PCR, then one of the following procedures may correct it.

1) Phenol Chloroform extraction using the protocol from July 24, 2013 with the following modification: 2μL of lysozyme added in the 3rd step with the 10% SDS, to encourage membrane disolution

2)Using the MoBio Power Soil DNA extraction kit and included protocol.

![HTML tutorial](https://static.igem.org/mediawiki/2013/7/7b/120.png)

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#

07/25/13

**Transformation of Ca Promoters in pSB1C3**

Most plates had growth. Plates are going to be incubated at 37°c for another night. Tomorrow a library will be made.

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#

07/26/13

**Transformation of Ca Promoters in pSB1C3**

Colonies were larger. Transformant libraries were made of Ca promoters in addition to ter, Adh6, and C. cell Adh. Plates were parafilmed and placed in 37°C incubator to prevent drying out over the weekend. Monday, plates will be streaked, Tuesday, plasmids will be extracted, and Wednesday, plasmids will be sent for sequencing.

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#

07/25/13

**PCR of BktB for Site-Directed Mutagenesis**

Repeated PCR of BktB using primers for sie-directed mutagenesis

Also included one PCR series using the Forward and Revers primers for the whole gene

Used only undiluted templates

Temperature gradient was selected to identify correct temperature for each pair of primers (assuming template is present)

Used wells:

1: T = 50&#176:c

4: T = 52.7&#176:c

6: T = 54.5&#176:c

8: T = 56.4&#176:c

12: T = 60&#176:c

![HTML tutorial](https://static.igem.org/mediawiki/2013/f/fe/118.png)

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#

07/25/13

**Gel of BktB Site-Directed Mutagenesis PCR**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample | Dilution || 1 | Empty |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Kb plus ladder || 3 | Forward + SDM1 Reverse | 1:10 || 4 | Forward + SDM1 Reverse | 1:100 || 5 | Forward + SDM1 Reverse | 1:1000 || 6 | Forward + SDM1 Reverse | 1:10000 || 7 | SDM1 Forward + SDM2 Reverse | 1:10 || 8 | SDM1 Forward + SDM2 Forward | 1:100 || 9 | SDM1 Forward + SDM2 Reverse | 1:1000 || 10 | SDM1 Forward + SDM2 Reverse | 1:10000 || 11 | SDM2 Forward + Reverse | 1:10 || 12 | SDM2 Forward + Reverse | 1:100 || 13 | SDM2 Forward + Reverse | 1:1000 || 14 | SDM@ Forward + Reverse | 1:10000 || 15 | Kb plus Ladder || 16 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/7/79/117.png)

No PCR products.

Will run a temperature gradient for each sample to see if temperature is affecting enzyme activity

Will preform Power Soil DNA Extraction and bead beaten with phenol: chloroform extraction and isopropanol precipitation to see if template is the problem.

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#

07/24/13

**Site-Directed Mutagenesis PCR for BktB**

Into each tube:

* Mix: 98μL
* Template: 2μL
* Total: 100μL

Dilutions used: 1:10, 1:100, 1:1000, 1:10000

Mix:

* 2x Master Mix: 250μL
* Forward Primer: 3μL
* Reverse Primer: 3μL
* PCR dH2O: 244μL
* Total: 500μL

PCR Primers Used:

* Forward + SDM1 Reverse

+ 340 base pairs

* SDM1 Forward + SDM2 Revers

+ 660 base pairs

* SDM2 Forward + Reverse

+ 300 base pairs

![HTML tutorial](https://static.igem.org/mediawiki/2013/d/d1/116.png)

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#

07/24/13

**DNA Extraction of R. eutropha**

1. Re suspend cells in 500μL 1x STE buffer
2. Vortex until mixture is homogenous
3. Add 50μL of 10% Sodium-dodecyl sulfate (SDS)
4. IF cells insufficiently lysed, 2 minutes in bead beater
5. Add 500μL of Chloroform:Phenol pH 8

* Extract bottom layer (2 phases present)
* Minimize time the chloroform is out of fridge

6. Vortex
7. Centrifuge for 30 seconds

* 14000 rpm

8. Transfer top phase to 1.5mL microcentrifuge tube

* Don't take up white precipitant from interphase of solution

9. Add 500μL of isoamyl acetate (CIAA 24:1) to top phase
10. Vortex
11. Centrifuge for 30 seconds

* 14000 rpm

12. Transfer top phase to 1.5mL microcentrifuge tube

* Don't take up white precipitant from interphase of solution

13. REPEAT STEPS 9-12

* Note: Keep track of amount finally transfered in step 12

14. Add (2/10) volume (amount of top phase) of 3M Sodium Acetate
15. Add (7/10) volume (step 14 amount) of Isopropanol

* Note: Mix solutions with pipette

16. Centrifuge for 25 minutes

* 14000 rpm

17. Dispose of liquid portion

* Don't disturb invisible pellet

18. Dry pellet on heat block at 60°C for 10 minutes

* Leave caps open for evaporation of isopropanol

19. Re suspend pellet in 50μL of distilled water

![HTML tutorial](https://static.igem.org/mediawiki/2013/e/e1/115.png)

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#

07/24/13

**Repeat of Transformation of Ca Promoters into Top 10 Cells**

Preformed as outlined on February 11, 2013

-Last time we used 2μL for transformation this time since our ligations will contain a maximum of 100ng of insert (recommended amount for transformation of top 10 cells), we are going to use the entire ligation volume (20μL)

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#

07/23/13

**Making Beef Extract Medium**

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Amount || Beef Extract | 3g || Peptone | 5g || Agar | 15g || Distilled Water | 1000mL ||

Pour components and one magnetic mixer into empty jar. Fill jar with distilled water to 1000mL mark.

Autoclave at setting number "2" for 20 minutes at slow exhaust

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#

07/23/13

**PCR of Ca AdhE**

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Amount || 2X Master Mix | 125μL || Forward Primer | 4μL || Reverse Primer | 4μL || PCR Water | 117μL || TOTAL | 250μL ||

1 Reaction 50μL = 49μL Mix + 1μL Template

![HTML tutorial](https://static.igem.org/mediawiki/2013/f/f7/110.png)

![HTML tutorial](https://static.igem.org/mediawiki/2013/2/29/110.1.png)

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#

07/23/13

**Digest of Cc Adh and Cs Adh**

500ng DNA for each reaction

500ng (1&#956L⁄39.7ng) = 12.5 μL Cc DNA

500ng (1&#956L⁄36.0ng) = 14 μL Cs DNA

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Cc | Cs | pSB1C3 || 10X Buffer | 2μL | 2μL | 2μL || EcoR1 | 2μL | 2μL | 2μL || Pst1 | 2μL | 2μL | 2μL || DNA | 12.5μL | 14μL | 2.5μL || PCR Water | 1.5μL | 0μL | 11.5μL || Total | 20μL | 20μL | 20μL ||

500ng (1&#956L⁄202ng) = 2.5μL pSB1C3 backbone

Incubate at 37°C water bath for 45 minutes

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#

07/11/13

**Repeat of Cohesive End CLoning of gBloeu Ca Promoters Ptb, AaDc, and thl into pSB1C3 Vector**

1. Digest

A. In 1 tube contain

* Backbone: 2μL
* EcoR1: 1μL
* Pst1: 1μL
* 10x Buffer: 2μL
* Water: 14μL
* Total: 20μL

B. In each 6 tubes combine

* gBloeu Resuspension: 10μL
* EcoR1: 1μL
* Pst1: 1μL
* 10x Buffer: 2μL
* Water: 6μL
* Total: 20μL

Incubate in 37°C water bath for 1 hour

Clean with QIAquick PCR Purification Kit

Nano drop

![HTML tutorial](https://static.igem.org/mediawiki/2013/9/91/107.png)

Aadc quantification appears to be good with a reading of 1.9ng/μL, but is the lowest limit of detection for the nano drop 1000, so we can't be sure

Ligation

Since the vector is 31.7 ng/μL and the protocol calls for 50ng of DNA and the concentration of insert might be 1.5 to 2.0ng/μL due to the need of 20ng.

* pSB1C3: 2μL
* Promoter: 13μL
* Buffer: 2μL
* Ligase: 1μL
* Water: 2μL
* Total: 20μL

Incubate at room temperature overnight

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#

07/23/13

**Gel of AdhE and AdhE1**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Lane Number | Sample | Dilution | Temperature(μ) || 1 | Ladder |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 2 | Ca AdhE | 1:100 | 53 || 3 | Ca AdhE | 1:100 | 50 || 4 | Ca AdhE | 1:100 | 56 || 5 | Ca AdhE | 1:100 | 60 || 6 | Ca AdhE | 1:10 | 50 || 7 | Ca AdhE | 1:10 | 53 || 8 | Ca AdhE | 1:10 | 56 || 9 | Ca AdhE | 1:10 | 60 || 10 | Ca AdhE | 1:1000 | 50 || 11 | Ca AdhE | 1:1000 | 53 || 12 | Ca AdhE | 1:1000 | 56 || 13 | Ca AdhE | 1:1000 | 60 || 14 | Empty || 15 | Empty || 16 | Empty || Row #2 || 1 | Ladder || 2 | Ca AdhE1 | 1:100 | 50 || 3 | Ca AdhE1 | 1:10 | 50 || 4 | Ca AdhE1 | 1:100 | 53 || 5 | Ca AdhE1 | 1:100 | 50 || 6 | Ca AdhE1 | 1:100 | 56 || 7 | Ca AdhE1 | 1:10 | 53 || 8 | Ca AdhE1 | 1:10 | 56 || 9 | Ca AdhE1 | 1:10 | 60 || 10 | Ca AdhE1 | 1:1000 | 50 || 11 | Ca AdhE1 | 1:1000 | 53 || 12 | Ca AdhE1 | 1:1000 | 56 || 13 | Ca AdhE1 | 1:1000 | 60 || 14 | Empty || 15 | Empty || 16 | Empty || | |

![HTML tutorial](https://static.igem.org/mediawiki/2013/3/3a/106.png)

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#

07/22/13

**PCR of Ca AdhE and AdhE1**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Dilution | 50° | 53° | 56° | 60° || Empty || Ca AdhE | 1:10 | ✓ | ✓ | ✓ | ✓ || Ca AdhE | 1:100 | ✓ | ✓ | ✓ | ✓ || Ca AdhE | 1:1000 | ✓ | ✓ | ✓ | ✓ || Empty || Ca AdhE1 | 1:10 | ✓ | ✓ | ✓ | ✓ || Ca AdhE1 | 1:100 | ✓ | ✓ | ✓ | ✓ || Ca AdhE1 | 1:1000 | ✓ | ✓ | ✓ | ✓ ||

![HTML tutorial](https://static.igem.org/mediawiki/2013/0/01/105.png)

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#

07/22/13

**PCR of Ca AdhE/AdhE1 using pSOL**

Used Qia Quick Prep Plasmid Extraction Kit

AdhE and AdhE1 are supposed to be located on pSOL plasmid using pSOL as template (1:10, 1:100, 1:1000)

![HTML tutorial](https://static.igem.org/mediawiki/2013/8/8a/103.2.png)

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#

07/22/13

**Plasmid Extraction of Ca pSOL**

Used Qia Quick Prep Plasmid Extraction Kit

![HTML tutorial](https://static.igem.org/mediawiki/2013/f/fe/103.1.png)

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#

07/11/13

**Dual Gel of mlsR Xformants**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row #1 || Well # | Sample || 1 | Ladder || 2 | mlsR 5 || 3 | mlsR 9 || 4 | mlsR 10 || 5 | mlsR 12 || 6 | mlsR 22 || 7 | mlsR 24 ||

![HTML tutorial](https://static.igem.org/mediawiki/2013/6/64/103.png)

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#

07/22/13

**Dual Digest of mlsR Xformants**

EcoR1 and Pst1 Dual Digest of mlsR colonies

5, 9, 10, 12, 22, 24

500ng (1&#956L⁄112.2ng) = 4.45 μL → 4.5 μL

500ng (1&#956L⁄124.5ng) = 4.02 μL → 4.0 μL

500ng (1&#956L⁄117.7ng) = 4.25 μL → 4.5 μL

500ng (1&#956L⁄162.0ng) = 3.09 μL → 3.0 μL

500ng (1&#956L⁄188.7ng) = 2.65 μL → 2.5 μL

500ng (1&#956L⁄206.4ng) = 2.42 μL → 2.5 μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Colony Number | EcoR1 | Pst1 | 10x Buffer | DNA | PCR dH2O | Total || 5 | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL || 9 | 2μL | 2μL | 2μL | 4.0μL | 10.0μL | 20μL || 10 | 2μL | 2μL | 2μL | 4.5μL | 9.5μL | 20μL || 12 | 2μL | 2μL | 2μL | 3.0μL | 11.0μL | 20μL || 22 | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL || 24 | 2μL | 2μL | 2μL | 2.5μL | 11.5μL | 20μL |

Incubated at 37°L for 45 minutes

Transformed to 80°L heat block for 1 hour

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#

07/22/13

**Transformation of Terminators/Promoters**

Transforming the following parts from the iGEM distribution for use in shuttle vector

BBa\_B1006 from 2013 Plate 3, Well 6D (in C3)

BBa\_Bl003 from 2013 Plate 3, Well 4P (in C3)

Punctured wells with pipette tip

Re suspended DNA in 10μL of PCR water

1μL of DNA added to 50μL of competent cells

100μL of cells  Used 2μL of DNA

Incubated on ice for 30 minutes

Heat shocked cells for 1 minutes

Incubated on ice for 5 minutes

Add 600μL of Psi broth

Incubated at 37°C with shaking for 2 hours

Also Xformed

* BBa\_K148012 Pveg
* BBa\_K823000 P?
* BBa\_K823002 P?
* BBa\_K823003 Pveg
* BBa\_K090504 ?

Plated 90μL of undiluted 1:10, 1:100, 1:1000 of BBa\_B1006 and BBa\_B1003

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#

07/22/13

**PCR Cleanup of Cs Adh PCR Products**

![HTML tutorial](https://static.igem.org/mediawiki/2013/0/03/99.png)

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#

07/22/13

**Plasmid Extraction of Xformants**

mlsR library colonies 5, 9, 10, 12, 22, 24

Extracted using Qiagen plasmid prep kit

![HTML tutorial](https://static.igem.org/mediawiki/2013/e/ec/98.png)

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#

07/19/13

**PCR of Ccel Adh**

Preformed PCR to amplify Adh out of C. cellulolyticum

![HTML tutorial](https://static.igem.org/mediawiki/2013/f/fc/97.png)

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#

07/19/13

**PCR of Cb SAdh**

Choose lower temperature (50°C) for annealing.

![HTML tutorial](https://static.igem.org/mediawiki/2013/e/eb/96.png)

Still have primer dimer, or primers may be sitting down at another place on the genomic DNA

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#

07/19/13

**Promoter and Transformation Trouble Shooting**

No colonies have grown on the plates incubated with transformed Top 10 cells with pSB1C3 and various promoters plated on July 16 2013. We did a nanodrop quantification of the ligationproducts.

![HTML tutorial](https://static.igem.org/mediawiki/2013/c/c9/95.png)

While the 260/280 ratios seem high, Dr. wawrick says that they aren’t troubling. However, the amount of DNA quantified is unrealistic. Since, we only started with 100ng of insert and 200ng of vector. We ran a gel to see if what we quantified is DNA and not RNA.

Well 1-Ladder

Well 2-Aadc – RBS

Well 3- thl + RBS

From this gel, it doesn’t look like we actually have any DNA (although there may be some really faint bands near the bottom). This means that the ligation didn’t actually occur (or we would have seen a band at 2000bp + 125-75bp). However, if the ligation didn’t work we would still expect to see 2 bands. One is the size of the vector and the other is the size of the insert. Perhaps, something went wrong in the phenol/chloroform extraction step.

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#

07/19/13

**Restreak of mlsR + pSB1C3 Top 10**

Transferred six colonies from library to individual plates. Numbers 5, 9, 10, 12, 22, and 24 from library.

PCR Purification of July 18, 2013 Cs

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#

07/18/13

**PCR of C sacch and Cs Adh**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1 || Lane Number | Sample | Dilution | Temperature (°C) || 1 | Ladder || 2 | Cs | Undiluted | 55 || 3 | Cs | Undiluted | 62 || 4 | Cs | Undiluted | 65 || 5 | Cs | 1:10 | 55 || 6 | Cs | 1:10 | 62 || 7 | Cs | 1:10 | 65 || 8-16 | Empty || Row 2 || Lane Number | Sample | Dilution | Temperature (°C) || 1 | Ladder || 2 | Cb | Undiluted | 55 || 3 | Cb | Undiluted | 62 || 4 | Cb | Undiluted | 65 || 5 | Cb | 1:10 | 55 || 6 | Cb | 1:10 | 62 || Cb | Cs | 1:10 | 65 || 8-16 | Empty ||

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#

07/18/13

**Gel of Isolated Plasmids**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1 || Lane Number | Sample || 1 | Ladder || 2 | pSB1C3 + mlsR1 || 3 | pSB1C3 + mlsR7 || 4 | pSB1C3 + mlsR13 || 5 | pSB1C3 + mlsR13|  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | | 6 | pSB1C3 + mlsR21 || 7 | pSB1C3 + mlsR23 || 8 | pSB1C3 + mlsR26 || 9 | puc19 + KIVD || |

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#

07/18/13

**Dual Digests of Isolated Plasmids (18 July)**

500ng of plasmid for digest

puc19 + KIVD

500ng X (μL/516ng) = 0.89μL

pSB1C3 + mlsR21

500ng X (μL/99.4ng) = 5.03μL

pSB1C3 + mlsR13

500ng X (μL/81.0ng) = 6.17μL

pSB1C3 + mlsR1

500ng X (μL/101.0ng) = 4.95μL

pSB1C3 + mlsR7

500ng X (μL/65.1ng) = 7.68μL

pSB1C3 + mlsR23

500ng X (μL/84.1ng) = 5.95μL

pSB1C3 + mlsR26

500ng X (μL/86.1ng) = 5.81μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Gene | EcoR1 | Pst1 | 10x Buffer | DNA | PCR Water | Total || KIVD | 2μL | 2μL | 2μL | 1μL | 13μL || mlsR21 | 2μL | 2μL | 2μL | 5μL | 9μL || mlsR13 | 2μL | 2μL | 2μL | 6μL | 8μL || mlsR1 | 2μL | 2μL | 2μL | 5μL | 9μL || mlsR7 | 2μL | 2μL | 2μL | 7.5μL | 6.5μL || mlsR23 | 2μL | 2μL | 2μL | 6μL | 8μL || mlsR26 | 2μL | 2μL | 2μL | 6μL | 8μL ||

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#

07/18/13

**Plasmid Prep of Transformed Top10 Cells with pSB1C3 + mlsR and puc19 + KIVD**

![HTML tutorial](https://static.igem.org/mediawiki/2013/7/77/89.png)

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#

07/17/13

**DNA Extraction from Ca, Cs, Cb, and Cc using MoBio Power Soil DNA Extraction Kit**

![HTML tutorial](https://static.igem.org/mediawiki/2013/9/9d/88.png)

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#

07/10/13

**C. acetobutylicum Shuttle Vector Design**

The following sequences have been verified:

Clos Ori BBa\_J238001

Pthl BBa\_J238002

Paadc BBa\_J238003

Ptb BBa\_J238004

mlsR BBa\_J238005

Use Terminators for test testing

Long artificial terminator (T>90%)

* BBa\_B1006
* 39 base pairs
* 2013 p3w6D in C3

Small artificial terminator (T = 85%)

* BBa\_B1002
* 34 base pairs
* 2013 p5w4D in Ak3

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#

07/10/13

**1% Agarose Gel Electrophoresis of 9 July PCR Products**

Procedure followed as on pg. 50

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1 || Lane | Sample | Dilution | Temperature (°C) || 1 | Ladder || 2 C. sacc | 1:10 | 50.8 || 3 | C. sacc | 1:100 | 50.8 || 4 | C. sacc | 1:1000 | 50.8 || 5 | mlsR | 1:10 | 50.8 || 6 | mlsR | 1:100 | 50.8 || 7 | mlsR | 1:1000 | 50.8 || 8 | C beij | 1:10 | 50.8 || 9 | C beij | 1:100 | 50.8 || 10 | empty || 11 | empty || 12 | empty || 13 | empty || 14 | empty || 15 | empty || 16 | empty || Row 2|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | Ladder || 2 | C. sacc | 1:10 | 52.8 || 3 | C. sacc | 1:100 | 52.8 || 4 | C. sacc | 1:1000 | 52.8 || 5 | mlsR | 1:10 | 52.8 || 6 | mlsR | 1:100 | 52.8 || 7 | mlsR | 1:1000 | 52.8 || 8 | C beij | 1:10 | 52.8 || 9 | C beij | 1:100 | 52.8 || 10 | empty || 11 | empty || 12 | empty || 13 | empty || 14 | empty || 15 | empty || 16 | empty || Row 3 || 1 | Ladder || 2 C. beij | 1:10 | 54.1 || 3 | C. beij | 1:100 | 54.1 || 4 | mlsR | 1:10 | 54.1 || 5 | mlsR | 1:100 | 54.1 || 6 | mlsR | 1:1000 | 54.1 || 7 | C. sacc | 1:10 | 54.1 || 8 | C. sacc | 1:100 | 54.1 || 9 | C. sacc | 1:1000 | 54.1 || 10 | empty || 11 | empty || 12 | empty || 13 | empty || 14 | empty || 15 | empty || 16 | empty || Row 2|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | Ladder || 2 | C. beij | 1:10 | 58.8 || 3 | C. beij | 1:100 | 58.8 || 4 | mlsR | 1:10 | 58.8 || 5 | mlsR | 1:100 | 58.8 || 6 | mlsR | 1:1000 | 58.8 || 7 | C. sacc | 1:10 | 58.8 || 8 | C. sacc | 1:100 | 58.8 || 9 | C. sacc | 1:1000 | 58.8 || 10 | empty || 11 | empty || 12 | empty || 13 | empty || 14 | empty || 15 | empty || 16 | empty || | | | | | |

[Back to top](#top)

#

07/9/13

**DNA Extraction for C sacc, C aceto, and C beij**

Procedure followed as on pg. 50

[Back to top](#top)

#

07/9/13

**Gradient PCR of C. scc, mlsR, and C. beij**

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Column Number | Temperature (°C) || 3 | 50 || 5 | 52.8 || 6 | 54.1 || 10 | 58.8 ||

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Lane Number | Sample || 1 | C sacc 1:10 || 2 | C sacc 1:100|  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 3 | C sacc 1:1000 || 4 | mlsR 1:10 || 5 | mlsR 1:100 || 6 | mlsR 1:1000 || 7 | C beij 1:10 || 8 | C beij 1:100 || |

Vf Preparation

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Amount || 2x Mastermix | 125μL || Forward | 2μL || Reverse | 2μL || PCR dH2O | 121μL || Total | 250μL ||

[Back to top](#top)

#

07/3/13

**Gel of (2 July) PCR Products**

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1 || Lane | Sample || 1 | Ladder || 2 C. sacc 1:10 || 3 | C. sacc 1:100 || 4 | C. sacc 1:1000 || 5 | C. sacc 1:10000 || 6 | Negative || 7 | mlsR 1:10 || 8 | mlsR 1:100 || 9 | mlsR 1:1000 || 10 | mlsR 1:10000 || 11 | Negative || 12 | ladder || 13 | empty || 14 | empty || 15 | empty || 16 | empty || Row 2|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | 1 | Ladder || 2 | C. sacc 1:10 || 3 | C. sacc 1:100 || 4 | C. sacc 1:1000 || 5 | C. sacc 1:10000 || 6 | Negative || 7 | C. beij 1:10 || 8 | C. beij 1:100 || 9 | C. beij 1:1000 || 10 | C. beij 1:10000 || 11 | empty || 12 | empty || 13 | empty || 14 | empty || 15 | empty || 16 | empty || | | |

[Back to top](#top)

#

07/2/13

**PCR of mlsR and Redo of C. sacc and C. beij PCR**

[Back to top](#top)

#

07/2/13

**Ralstonia eutropha DNA Extraction**

Due to previous low extraction efficiency

Attempted to extract higher [DNA]

1. For 5mL tubes, each containing 3mL of meat extract broth inoculated with R. eutropha cells, which were in stationary phase
2. 1mL aliquots transferred stepwise to two 15mL epi tubes, spinning at 14,000 rpm for five minutes to pellet cells
3. Supernatant discarded and additional 1mL aliquot added, spun until all cells pelleted
4. Cells re suspended in 500μL if 1x STE buffer in each of the two epi tubes
5. Vortex
6. 50μL of 10% SDS added to each tube
7. 500μL of TE saturated phenol pH8 added to each tube
8. Vortex well and centrifuge for 30 seconds
9. Aqueous recovered
10. Phenol extraction
11. Steps 7-9 repeated
12. 500μL chloroform added to each tube and vortexed
13. Aqueous discarded
14. Chloroform extraction repeated- - 400μL split into each of four tubes
      - 100μL of 3M NaOH added to each tube
      - 350μL isopropanol added to each tube
      - Centrifuge at 14000 rpm for 25 minutes
      - Liquid discarded
      - Tubes places on 80°C heating block for 15 minutes
      - Contents of each tube re suspended in 500μL of PCR dH2O

      [Back to top](#top)

      #

      07/2/13

      **Gel Electrophoresis of PCR Product (1 July)**

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Lane | Sample || 1 | Ladder || 2 C. sacc Undiluted || 3 | C. sacc 1:10 || 4 | C. sacc 1:100 || 5 | Negative || 6 | C. cell undiluted || 7 | C. cell 1:10 || 8 | C. cell 1:100 || | |

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Lane | Sample || 1 | Ladder || 2 Negative Control || 3 | 1:10 Diluted C. beij || 4 | C. beij 1:100 || 5 | C. beij 1:1000 || 6 | C. beij 1:10000 || 7 | Empty || 8 | Empty || | |

      [Back to top](#top)

      #

      07/1/13

      **16S Gel Repeat**

      16S gel of C. acetobutylicum, R. eutropha, C. sacchaolyticum, and C. beijerinki to confirm wheather or not we have what we think we have

      |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | 16S Gel || Lane Number | Sample || 1 | Ladder || 2 | C. beijerinki || 3 | R. eutropha || 4 | C. sacchrolyticum || C. acetobutylicum ||

      [Back to top](#top)

      #

      06/28/13

      **1:4 Loading Dye**

      1 part loading dye

      2 parts 40% glycerol

      2 parts PCR DI water

      [Back to top](#top)

      #

      06/27/13

      **DNA Extraction of R. eutropha, C. saccharolyticum, and C. beijerinki**

      Procedure performed as outline on pg. 42

      16S PCR of C. acetobutylicum, R. eutropha, C. sacchrolyticum, and C. beijerinki to confirm wheather or not we have what we think

      |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | PCR Solution || Component | Amount (μL) || 2x PCR Mastermix | 350 || Forward primer (27F) | Reverse primer (1525R || PCR water | 344 || Total | 700 ||

      50μL for each reaction, three reactions for each organism with different template dilutions
      * Undiluted
      * 1:10
      * 1:100

      Place 1μL template into each tube

      Negative control won’t have a template

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Row Number | Colum Number | Sample || 3 | 7 | Negative Control || 4 | 4 | C. beijerinki || 4 | 5 | C. beijerinki 1:10 || 4 | 6 | C. beijerinki 1:100 || 4 | 7 | C. sacchrolyticum || 4 | 8 | C. sacchrolyticum 1:10 || 4 | 9 | C. sacchrolyticum 1:100 || 5 | 4 | C. acetobutylicum || 5 | 5 | C. acetobutylicum 1:10 || 5 | 6 | C. acetobutylicum 1:100 || 5 | 7 | R. eutropha || 5 | 8 | R. eutropha 1:10 || 5 | 9 | R. eutropha 1:100 ||

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Gel of PCR product || Row Number | Colum Number | Sample || 1 | 1 | Ladder || 1 | 2 | C. beijerinki || 1 | 3 | C. beijerinki 1:10 || 1 | 4 | C. beijerinki 1:100 || 1 | 5 | C. sacchrolyticum || 1 | 6 | C. sacchrolyticum 1:10 || 1 | 7 | C. sacchrolyticum 1:100 || 2 | 1 | Ladder || 2 | 2 | C. acetobutylicum || 2 | 3 | C. acetobutylicum 1:10 || 2 | 4 | C. acetobutylicum 1:100 || 2 | 5 | R. eutropha || 2 | 6 | R. eutropha 1:10 || 2 | 7 | R. eutropha 1:100 || 2 | 8 | Negative Control ||

      We have bands of the correct length for Cb, Cs, and Ca, they are too faint for sequencing. So, we are going to redo the PCR without template dilutions and do 35 cycles instead of 30 cycles

      [Back to top](#top)

      #

      06/25/13

      **Modified VM Medium**

      C. cellulolyticum may be grown at 37°L

      VM medium to reduce precicpitation

      AEM: 77, 2727-2733 (2011)

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Modified ? (per liter) || Component | Amount || KH2PO4 | 1g || KH2PO4 | 3.4g || Urea | 2.14g || MgCl2 - 6H2O | 1g || CaCl2 - 2H2O |0.15g| FeSO2 - 6H2 | 1.25g || MOPS 3-(N-morpholino)propanesulfuric acid | 10g || Rezazurin | 2mg || Vitamin solution | 10mL || Yeast Extract | 2g || Oligoelement solution | 1mL || Cysteine – HCl | 1g || Cellobiose | 5.1345 ||

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | 100x Vitamin Solution || ComponentAmount || Biotin | 0.08μM || Pyridoxamine | 0.02μM || Cyanocobalamine | 0.001μM|  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | | p-aminobenzoic acid | 0.15μM || Thiamine | 0.9μM || L-alaning | 0.22μM || |

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | 1000x Oligoelement Solution (per liter) || Component | Amount || FeSO4 - 7H2O | 5g || ZnSO4 - 7H2O | 1.44g || MnSO4 - 7H2O | 1.12g || CuSO4 - 5H2O | 0.25g || Na2B4O7 | 0.2g || (Mo)7(NH4)6O24 - 4H2O | 1g || NiCl2 | 0.04g || CoCl2 | 0.02g || HBO3 | 0.03g || Na2SeO3 | 0.02g || 10M HCl | 50mL ||

      VM Medium placement

      * 100mL in bottles
      * 20mL in vials[Back to top](#top)

      #

      06/25/13

      **Re constituting Strains in Anaerobic Chamber**

      * Clean glass vial with acetone
      * In aerobic Chamber:
      + Chemwipes
      + Sterile Needles* Bring into chamber
      + Heavy duty aluminum foil to work over
      + Glass cutter
      + File
      1. Cycle chamber airlock; twice
      2. Clean gloves and chamber with 1N HCl (x2)
      3. ???????????????????
      4. Use sterile syringe to add 0.5mL media to inner vial
      5. Mix with syringe
      6. Add 0.5 of inoculums to each of the two media vials
      7. Transfer an inoculum from these vials to a second vial to get one heavy and one light inoculum
      [Back to top](#top)

      #

      06/24/13

      **Media Preparation for C. acetobutylicum**

      Media components were added to a 2000mL Erlenmeyer flask

      0.1mL of 1g/L Rezarin was added to the medium and was degassed under N2 for 45 minutes

      [Back to top](#top)

      #

      06/21/13

      **Media Preparation for C. acetobutylicum**

      C. acetobutylicum will grow on defined media:

      AEM 50:1238 (1985) or the following

      |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Dr. Tanner’s recipe of C .acetobutylicum (per liter) || Component | Amount || Yeast Extract | 1g || Glucose/Dextrose | 20g || RST minerals | 10mL || RST vitamins | 10mL || RST metals | 5mL ||

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | RST Minerals (per liter) || Component | Amount || NaCl | 80g || NH4Cl | 100g || KCl | 10g || KH2PO4 | 10g || MgSO4 - 7H2O | 20g || CaCl2 - 2H2O | 4g ||

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | RST Minerals (per liter) || Component | Amount || Nitriloacetic Acid | 2g || MnSO4 - H2O | 1g || Fe(NH4)2(SO4)2 - 6H2O | 0.8g || CoCl2 - 6H2O | 0.2g || 2nSO4 - 7H2 | 0.2g || CuCl2 - 2H2O | 0.02g || NiCl2 - 6H2O | 0.02g || Na2MoO4 - 2H2O | 0.02 || Na2SeO4 | 0.02g || Na2WO4 | 0.02g ||

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | RST Vitamins || Components | Amount | Light Sensitivity (Y/N) || Pyridoxine HCl | 10mg | Y || Thiamine HCl | 5mg | Y || Riboflavin | 5mg | Y || Calcium Pantothenate | 5mg | Y || Thioctic Acid | 5mg | N || p-Aminobenzoic Acid | 5mg | Y || Nicotinic Acid | 5mg | N || B12 = Cyanocobalamine | 5mg | N || Biotin | 2mg | N || Folic Acid | 2mg | Y || Mercaptopethane Sulfuric Acid | 10mg | N ||

      [Back to top](#top)

      #

      06/17/13

      **Competent pAN1 containing DH5 E. coli**

      Suspended a unit sized inoculums of pAN1 containing DH5 E. coli in LB with chloramphenicol resistance.

      Incubated overnight at 37°C and at 220 rpm for 2.5 hours

      One culture has an OD600 of 0.38 and another is 0.475

      * Spun at 12000 g for 20 minutes
      * Discarded supernatant and re suspended in 15mL of TFBl
      * Incubated on ice for 60 minutes
      * Centrifuged at 12000 g for 20 minutes
      [Back to top](#top)

      #

      06/14/13

      **Gel of puc19 with KIVD Digest (EcoR1 and Pst1)**

      [Back to top](#top)

      #

      06/14/13

      **Digestion of pUC19 containing KIVD with EcoR1-Hf and Pst1-Hf**

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Component | Amount (μL) || EcoR1 | 1 || Pst1-Hf | 1 || 10x Buffer | 2 || DNA | 2.5 (500ng) || PCR H2 | 13.5 || Total | 20 |

      Place in 37°C water bath for 45 minutes

      [Back to top](#top)

      #

      06/13/13

      **Gel of PCR**

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Row 1 || Well Number | Sample | Temperature (°C) || 1 | Ladder || 2 | AdhE1 | 50.8 || 3 | AdhE1 | 52.8 || 4 | AdhE1 | 54.1 || 5 | AdhE1 | 58.8 ||

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Row 2 || Well Number | Sample | Temperature (°C) || 1 | Ladder || 2 | AdhE | 50.8 || 3 | AdhE | 52.8 || 4 | AdhE | 54.1 || 5 | AdhE | 58.8 ||

      The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

      AdhE = 2589 base pairs

      AdhE1 = 2577 base pairs

      [Back to top](#top)

      #

      06/12/13

      **PCR redo of AdhE and AdhE1**

      Temperature gradient and dilution up to 1:10000

      |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Column Number | Temperature (°C) || 3 | 50.8 || 5 | 52.8 || 6 | 54.1 || 10 | 58.8 ||

      [Back to top](#top)

      #

      06/12/13

      **Plasmid Isolation of pUC19 from BBa\_k273006**

      [Back to top](#top)

      #

      06/10/13

      **Amplification of AdhE and AdhE1 in C. acetobutylicum**

      Testing our primers using the template prepared on June/4

      Forward primer used (AdhE)

      Forward primer sequence

      Reverse primer used (AdhE)

      Reverse primer Sequence

      Forward primer used (AdhE1)

      Forward primer sequence

      Reverse primer used (AdhE1)

      Reverse primer sequence

      Preparation of solution 1:

      Into two separate 1.5mL centrifuge tubes

      |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Component | Amount (μL) || PCR DI Water | 146 || 2x PCR Mastermix | 150 || Forward primer | 2 || Reverse primer | 2 || Total | 300 ||

      50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)

      Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000

      1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1

      The 5th PCR tube containing solution 1 will serve as negative control

      [Back to top](#top)

      #

      06/4/13

      Place pxy plates in glove box to degrass

      Extracted Ca DNA

      1. Re suspend cells in 500μL 1x STE buffer
      2. Vortex
      3. Add 1/10 volume of 10% SDS
      4. 2 minutes in bead beater
      5. Add 500μL TE saturated phenol pH 8
      * Be sure to go down far enough in bottle to reach phenol
      * Put directly back in fridge when done6. Vortex
      7. Centrifuge for 30 seconds
      8. Recover aqueous phase
      * Don’t recover white material at interphase
      * Throw away9. Add 500μL Chloroform: Isoamyl acetate
      10. Vortex
      11. Centrifuge for 30 seconds- Recover and throw away aqueous phase
          - Repeat Steps 8-12- - Add 2/10 volumes 3M sodium acetate
              - Add 7/10 volumes isopropanol
              - Mix well
              - Centrifuge for 25 minutes
              - Remove liquid and don’t disturb invisible pellet
              - Dry pellet at 60°C in heat block for 10 minutes
              * Leave caps off to allow isopropanol to evaporate- Re suspend pellet in 50μL of dH2

      [Back to top](#top)

      #

      05/31/13

      **Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin**

      Digestion as preformed on pg. 47

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Gel of Digestion || Row 1 || Well Number | Sample || 1 | Ladder || 2 | 1 || 3 | 3 || 4 | 5 || 5 | 8 || 6 | 11 || 7 | 12 || Row 2 || Well Number | Sample || 1 | Ladder || 2 | 16 || 3 | 18 || 4 | 20 || 5 | 22 || 6 | 24 ||

      Wells 2 and 3 like like what we need.

      Sequencing primers are in the process of being made to comfirm

       2 |AdhE1 50.8 || 3 |AdhE1 52.8 || 4 |AdhE1 54.1 || 5 | AdhE1 |58.8|

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Row 2 || Well Number | Sample | Temperature (°C) || 1 | Ladder || 2 | AdhE | 50.8 || 3 | AdhE | 52.8 || 4 | AdhE | 54.1 || 5 | AdhE | 58.8 ||

      The gel results of this PCR are similar to the last one. The bands indicate smaller than 250 base pairs, which is smaller than the size of the genes we intended to amplify (2000 base pairs).

      AdhE = 2589 base pairs

      AdhE1 = 2577 base pairs

      [Back to top](#top)

      #

      06/12/13

      **PCR redo of AdhE and AdhE1**

      Temperature gradient and dilution up to 1:10000

      |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Column Number | Temperature (°C) || 3 | 50.8 || 5 | 52.8 || 6 | 54.1 || 10 | 58.8 ||

      [Back to top](#top)

      #

      06/12/13

      **Plasmid Isolation of pUC19 from BBa\_k273006**

      [Back to top](#top)

      #

      06/10/13

      **Amplification of AdhE and AdhE1 in C. acetobutylicum**

      Testing our primers using the template prepared on June/4

      Forward primer used (AdhE)

      Forward primer sequence

      Reverse primer used (AdhE)

      Reverse primer Sequence

      Forward primer used (AdhE1)

      Forward primer sequence

      Reverse primer used (AdhE1)

      Reverse primer sequence

      Preparation of solution 1:

      Into two separate 1.5mL centrifuge tubes

      |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Component | Amount (μL) || PCR DI Water | 146 || 2x PCR Mastermix | 150 || Forward primer | 2 || Reverse primer | 2 || Total | 300 ||

      50μL of solution 1 were pipette into 5 PCR tubes for each solution (AdhE and AdhE1)

      Template dilutions were prepared for 1:10, 1:100, 1:1000, and 1:10000

      1mL of template was loaded into 2 each of 4 PCR tubes containing 50μL of solution 1

      The 5th PCR tube containing solution 1 will serve as negative control

      [Back to top](#top)

      #

      06/4/13

      Place pxy plates in glove box to degrass

      Extracted Ca DNA

      1. Re suspend cells in 500μL 1x STE buffer
      2. Vortex
      3. Add 1/10 volume of 10% SDS
      4. 2 minutes in bead beater
      5. Add 500μL TE saturated phenol pH 8
      * Be sure to go down far enough in bottle to reach phenol
      * Put directly back in fridge when done6. Vortex
      7. Centrifuge for 30 seconds
      8. Recover aqueous phase
      * Don’t recover white material at interphase
      * Throw away9. Add 500μL Chloroform: Isoamyl acetate
      10. Vortex
      11. Centrifuge for 30 seconds- Recover and throw away aqueous phase
          - Repeat Steps 8-12- - Add 2/10 volumes 3M sodium acetate
              - Add 7/10 volumes isopropanol
              - Mix well
              - Centrifuge for 25 minutes
              - Remove liquid and don’t disturb invisible pellet
              - Dry pellet at 60°C in heat block for 10 minutes
              * Leave caps off to allow isopropanol to evaporate- Re suspend pellet in 50μL of dH2

      [Back to top](#top)

      #

      05/31/13

      **Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin**

      Digestion as preformed on pg. 47

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Gel of Digestion || Row 1 || Well Number | Sample || 1 | Ladder || 2 | 1 || 3 | 3 || 4 | 5 || 5 | 8 || 6 | 11 || 7 | 12 || Row 2 || Well Number | Sample || 1 | Ladder || 2 | 16 || 3 | 18 || 4 | 20 || 5 | 22 || 6 | 24 ||

      Wells 2 and 3 like like what we need.

      Sequencing primers are in the process of being made to comfirm

      [Back to top](#top)

      #

      05/30/13

      **Plasmid Extraction/Purification of Top 10 E. coli Transformed with pSB1C3 and Clostridial Origin**

      Purified six tubes of DNA (plasmid)

      Quantification with Nanodrop

      |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Digestion || Component | Amount (μL) || EcoR1 | 2 || Pst1 | 2 || 10x Buffer | 1 ||DNA 5 ||

      Incubate in 37°C water bath for 1 hour

      Load all 10μL onto gel

      |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
      | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
      | Gel of Digestion || Well Number | Sample || 1 | Ladder || 2 | Screen #3 || 3 | Screen #6 || 4 | Screen #10 || 5 | Screen #19 || 6 | Screen #19 || 7 | Screen #26 ||

      Note: Screen # corresponds to quantification Id

      Since our insert is about 700 base pairs and our vector is about 2000 base pairs. Well 2 looks like what we want. We are going to re streak colonies isolated from plate with screen #3 , make a freezer stock, and take more colonies from screen #10

      Freezer stocks of Top 10, pAN1, pIKM1, and p34KM were made by suspending cells in 1mL of 10% glycerol.

      [Back to top](#top)

      #

      05/29/13

      Made 80 stocks of C. acetobutylicum (2mL of culture into 2mL of glycerol)

      Streaked three more Top 10 pSB1C3 and Clostridial Origin plates

      [Back to top](#top)

      #

      05/28/13

      Inoculated cultures of C. acetobutylicum and a transfer was made

      Six plates (LB + Chloramphenicol) were streaked with transformation of pSB1C3 and Clostridial Origin in Top 10 E. coli cells in preparation for plasmid extraction.

      [Back to top](#top)

      #

      05/23/13

      b>

After 824: possible growth
[Back to top](#top)

#

05/22/13

**Re Dual Digest of pSB1C3 and Insert**

Procedure as preformed on pg. 38

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Gel of pSB1C3 and Insert Digestion || Well Number | Sample || 1 | Ladder || 2 | pSB1C3 || 3 | Clostridial Origin ||

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Gel of pSB1C3 and Insert Digestion after Quanitification || Well Number | Sample || 1 | Ladder || 2 | pSB1C3 || 3 | Clostridial Origin ||

Ligation:

* Insert, Vector: 21μL
* Ligase Buffer: 5μL
* Ligase: 3&#956:L

Let reaction sit on bench for 90 minutes

OR

For overnight ligation: 14°C water bath

[Back to top](#top)

#

05/9/13

**Repeat of Dual Digest of pSB1C3 and Clostridial Origin Fragment**

We’re repeating this procedure, due to that fact that days between a digestion and ligation shows to be ineffective. Hence, a ligation should be completed soon after digestion.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| pSB1C3 || Component | Amount (μL) || EcoR1 | 2 || Pst1 | 2 || 10x Buffer | 2 || DNA | 2.5 || PCR H2 | 11.5 || Total | 20 ||

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Clostridial Origin Fragment || Component | Amount (μL) || EcoR1 | 2 || Pst1 | 2 || 10x Buffer | 2 || DNA | 10 || PCR H2 | 4 || Total | 20 ||

Place in 37°C water bath for 15 minutes

Ran gel

 Well Number | Sample || 1 | Ladder || 2 | Clostridial Origin || 3 | pSB1C3 ||

Used wrong kit (Spin mini prep). Suppose to use PCR purification kit.

\

Cleaned/purified digests

Clostridial Origin

pSB1C3

Ligation:

* 5μL 10x Buffer
* 2μL ligase (quick)
* ?μL vector
* ?μL insert
* Up to 50μL dH2

[(ng vector X size insert in Kb)/Size vector in Kb] X molar ratio of insert : vector = ng of insert

Since we used the wrong kit, we’re going to precipitate the DNA out (used digestion on May/9)

Phenol Chloroform Protocol pH 8:

* Turn on heating block
* Add equal amounts of phenol and whatever volume DNA is in (20μL)

+ Phenol : Pipette below top surface

* Vortex
* Centrifuge for 30 seconds

+ Two layers appear
+ Protein is inactive and at interphase+ + Pipette out top layer of both and combine aqueous
    - Need to get rid of phenol because it absorbs same wavelength as does water and interferes with enzyme activity

* Add 50μL of CIAA 24:1

+ Removes Phenol

* Vortex
* Centrifuge for 30 seconds
* Remove top layer (aqueous)

+ Leave a little behind so not to contaminate aqueous phase with bottom layer

* Add 50μL of CIAA 24:1
* Vortex
* Centrifuge for 30 seconds
* Remove top layer (aqueous)

+ Leave a little behind so not to contaminate aqueous phase with bottom layer

Now we have 50μL of DNA

* Add 1/10 volume sodium acetate 3M pH 5.2
* Add 7/10 volume (total) of isopropanol
* Vortex
* Centrifuge for 20 minutes at 14,000 rpm
* Dispose of liquid, leaving pellet inside tube
* Add 50μL of 10% ethanol to get rid of isopropanol
* Centrifuge for 30 seconds
* Dispose of liquid
* Use heat block to dry

+ 60°C for 15 minutes
+ Leave tube open

* Add 10μL of PCR water to re suspend pellet
* Use 2μL to quantify via nandrop
* Add 1μL Ligase (quick)
* Add 1μL 10x Buffer = 10μL reaction
* Let reaction sit at room temperature for 2 hours

[Back to top](#top)

#

05/9/13

**Dual Digest of pSB1C3 and Clostridial Origin Fragment**

pSB1C3 = 2000 base pairs

Costridial Origin = 700 base pairs

Want to digest 500ng of backbone and 1:1 ratio of backbone to insert

pSB1C3 linearized backbone = 202.1 ng/μL

500ng of pSB1C3:
 500ng x μL/202ng = 2.5μL

1:1 mole ratio of backbone to insert

 500ng x (2000 bp/mol /700 bp/mol) = 175ng insert

175ng of insert:

 175ng x μL/17ng = 10μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| pSB1C3 || Component | Amount (μL) || EcoR1 | 2 || Pst1 | 2 || 10x Buffer | 2 || DNA | 2.5 || PCR H2 | 11.5 || Total | 20 ||

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Clostridial Origin Fragment || Component | Amount (μL) || EcoR1 | 2 || Pst1 | 2 || 10x Buffer | 2 || DNA | 10 || PCR H2 | 4 || Total | 20 ||

Place in 37°C water bath for 15 minutes

[Back to top](#top)

#

05/9/13

**PCR Cleanup of Linearized Backbone of Quantification**

Used QIAquick PCR Cleanup Kit and processed pSB1K3 and pSB1A3 linearized plasmid backbone PCR products

Nanodrop Quantification

[Back to top](#top)

#

05/9/13

**Gel of New Linearized Backbone using Provided Supermix**

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Well Number | Sample || 1 | Ladder || 2 | pSB1K3 (blue Hp) (fail: pipette tip fell into well) || 3 | pSB1K3 || 4 | pSB1A3 (blue Hp) || 5 | pSB1A3 ||

[Back to top](#top)

#

05/8/13

**Linearized Backbone PCR**

Dilute plasmid backbones to 10ng/μL

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Two reactions for pSB1K3 and pSB1A3 || Component | Amount (μL) || PCR High Fidelity Supermix | 97 || Primer 1: SB prep 3P-1 | 1 || Primer 2: SB prep 2Ea | 1 || Template | 10ng ||

pSB1K3

 10ng x μL/75ng = 1 : 7.5μL Dilution 10μL : 65μL PCR H2O

pSB1A3

 10ng x μL/40ng = 1 : 4μL Dilution 10μL : 30μL PCR H2O

Thermo Cycler (x38)

* 95°C for 2 minutes
* 95°C for 30 seconds
* 55°C for 30 seconds
* 68°C for 3 minutes
* 69°C for 10 minutes

[Back to top](#top)

#

02/12/13

stuff stuff stuff stuff

[Back to top](#top)

#

05/1/13

**PCR Supermix High Fidelity Preparation**

To have eight reaction vials with 50μL supermix each. Extra is made for potential pipetting error.

Component | Amount (μL) || Amplification Buffer 10x | 50 || dNTP Mixture (10 mM) | 15 || 50 mM MgSO4 | 10 || Pfx DNA Polymerase (Hf) (Spin down) | 10 || dH2O | 415 || Total (two different backbones) | 500 ||

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PCR reaction of linearized backbone in each tube || Component | Amount (μL) |
 PCR Supermix HF | 50 || SB prep 3P-1 | 0.4 || SB prep 2Ea | 0.4 || DNA | 5ng ||

pSB1K3

 5ng x μL/28ng =0 .18μL => 0.2μL

pSB1A3

 5ng x μL/135.8ng& = 0.037μL => 0.04μL

For our eight reactions, multiply all values (except supermix) above by 10 and add/mix into 250μL of supermix

 Final Reaction Tube Composition || Component | Amount (μL) || PCR Supermix | 250 || SB prep 3P-1 | 4 || SB prep 2Ea | 4 || pSB1K3 DNA | 50ng = 2μL || pSB1A3 DNA | 50ng = 0.4μL ||

Alliquot 55μL of supermix , primers, and template into each tube

To prepare dNTP mix

* 10μL of each NYP
* 60μL of dH2O

PCR for linearized backbone (actual)

Four reaction of 1 backbone || Component | Amount (μL) || 10x Buffer | 25 || Polymerase Hf (spin) | 5 || Mg | 5 || dNTP mix | 7.5 || DI H2O | 203 || Forward Primer | 2 || Reverse Primer | 2 || Template | 1 (may need to dilute) || Total | 250 ||

To dilute pSB1A3:(135.8 ng/μL)

1μL Template

4μL PCR dH2O

We didn’t get what we were trying to amplify

[Back to top](#top)
[Back to top](#top)

#

04/28/13

**PCR Reaction with Primer Set #3**

Preformed as on pg. 15 with the following modifactions

* Dilutions
  + 1:10
  + 1:100
  + 1:1000

1. 95°C for 45 seconds
2. 52°C for 45 seconds
3. 72°C for 2 minutes

Repeat 30x

32ng/μL of DNA template (pIKM1) successfully produced the Clostridial origin

PCR product was digested using QIAquick PCR purification kit

Note: If solution turns pick, the pH needs to be adjusted with 5μL of sodium acetate

[Back to top](#top)

#

04/24/13

**Anaerobic Media Preparation**

For 0.5L

* 500mL dH2
* 15g TSB mix
* 1g glucose* * Adjust pH to 7.3
    * Place sponge stopper in place
    * Open silver valve and black valve
    * Set degassing station to 20 psi
    * Switch to nitrogen and run for 30 seconds to flush oxygen out of head space
    * Place largest needle into media and turn on spin located on stir-plate, then set time for 45 minutes
    + Disinfect top of bottle with alchol wipe
    + Add sterile wipe filter with new needle
    + Need steady stream of bubbles* Add 0.1mL Resasrin (will be purple until autoclaved)
    * Post autoclaved
    + Pink color signifies media has been exposed to oxygen
    + No color in media signifies media is anaerobic* Oxygen scavenger
    * If black precipitant forms after autoclave and before transfer into media bottles, then too much oxygen present.

* Use acid washed vials
* Place stoppers in ice and water to shrink pores
* Flush bottles and vials with nitrogen
* Place 35mL in each using automated pipette
* Angle stopper with needle (gas still within)
* Leave for 10 seconds
* Bring needle out of bottle and push stopper in
* Crimp top
* Add 15mL to vials
* Flush head space

+ Put regular needle in and flushing needle in
+ Flush for 3 minutes
+ Pull both needles out at same times so you don’t put pressure on tubes

OR

+ Switch degassing station to vacuum
+ Alternate between vacuum and pressure 10 times

- Let it settle back to position before going back to pressure

+ Make sure all needles are in black stopper
+ Tubes now have 20 psi and poke with needle to vent
+ Turn off tank
+ Autoclave immediately

[Back to top](#top)

#

04/19/13

**PCR of Clostridial Origin with New Primers**

1. 95°C for 45 seconds
2. 52°C for 45 seconds
3. 72°C for 2 minutes

Repeat 30x

+ well 1: 1:10 dilution of template
+ well 2: 1:100 dilution
+ well 3: 1:1000 dilution
+ well 4: negative control

The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had

[Back to top](#top)

#

04/19/13

**Making Bulk Amounts of Linearized Plasmid Backbones**

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PCR Supermix High Fidelity | 9.6mL|  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | | Primer SB-prep 2Eb | 65μL || Primer SB-prep 3P-1 | 65μL || DNA Template | 100ng || |

pSB1K3

 100ng x μL/40ng = 9.8μL => 2.5μL + 7.5μL dH2O

pSB1A3

 100ng x μL/75ng& = 1.6μL => 1.5μL + 8.5μL dH2O

pSB1C3

 100ng x μL/43ng& = 1.6μL => 2.5μL + 7.5μL dH2O

High fidelity aliquots of 100μL for PCR

PCR

1. 95°C for 2 minutes
2. 95°C for 30 seconds
3. 55°C for 30 seconds
4. 68°C for 3 minutes
5. 68°C for 10 minutes

PCR Cleanup: Use Quiagen

[Back to top](#top)

#

04/9/13

**Gel of 8 Apr 2013 Digest Purifications**

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1 || Lane | Sample || 1 | 1 Kb Plus Ladder || 2 | pIKM1 + RSA1 digest || 3 | pIKM1 || 4 | pAN1 + RSA1 digest || 5 | pAN1 ||

85 Volts for 1 hour

[Back to top](#top)

#

04/8/13

**Verification of pIKM1/pANi**

Because we may have accidently stimulated the plasmid and mislabeled them previously. Since, no digestion of the plasmid pIKM1 from last weeks experiment is consistent with a methylating plasmid, today we are digesting both plasmids and running a gel.

This should verify that either we mixed up our plasmids, or we received the wrong plasmid.

1) Plasmid preparation of pAN1 and pIKM1 via Qiagen QiaPrep Spin Miniprep Unit instructions

2) Nanodrop quantification of plasmid DNA

3) Digest of pIKM1 and pAN1 with RSA1

pAN1

 500ng;times;μL/51ng = 9.8μL => 10.0μL

pIKM1

 500ng;times;μL/320ng& = 1.6μL => 2μ

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Compnents | pAN1 | pIKM1 || DNA | 10μL | 2μL || RSA1 | 2μL | 2μL || 10x Buffer | 2μL | 2μL || PCR Water | 6μL | 14μL || TOTAL | 20μL | 20μL ||

Placed in 42°C water bath for 15 minutes

[Back to top](#top)

#

04/5/13

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Row 1 || Lane | Sample || 1 | 1 Kb Plus Ladder || 2 | pSB1C3 + RSA1 + Xba + Invitrogen Buffer || 3 | pSB1C3 + RSA1 + Xba + Fermentas Buffer || 4 | pSB1C3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer || 5 | pSB1A3 + RSA1 + Xba + Invitrogen Buffer || 6 | pSB1A3 + RSA1 + Xba + Fermentas Buffer || 7 | pSB1A3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer || 8 | PIKM1 + RSA1 || 9 | pSB1K3 + Hind1 + Xba || 10 | Negative Control || 11 | PIKM1 || 12 | pSB1K3 ||

[Back to top](#top)

#

04/3/13

Since we couldn’t find information about the Fermentas buffer needed for Xba1 and Hind3, we are going to set up our experiment in such a way that tests to see if using only the buffer from Fermentas, using the buffer from Invitrogen, or using a 1:1 mixture of the two will allow our digestion to occur in reactions where enzymes from different companies are being used.

Tubes will be labeled with the plasmid name, which enzyme used, and which buffer was used. For example

+ pSB1C3
+ RSA1 and Xba1
+ 2μL of Invitrogen buffer

+ pSB1C3
+ RSA1 and Xba1
+ 2μL of Fermentas buffer

+ pSB1C3
+ RSA1 and Xba1
+ 1μL of Fermentas buffer and 1μL of Invitrogen buffer

We incubated digests in 42°C water bath for 15 minutes

[Back to top](#top)

#

04/1/13

**Restriction Digest of Biobrick and PIKM1 with AluI**

PPIKM1

+ 5μL Plasmid
+ 2μL RSA1
+ 2μL 10x Buffer
+ 11μL PCR Water

Biobrick

+ 5μL Plasmid
+ 2μL RE1(XbaI)
+ 2μL RE2(Hind 3 or RSA1
+ 2μL 10x Buffer
+ 9μL PCR Water

Use a gel of 2% agarose

+ 0.8g of agarose
+ 40mL of TAE 1x

Regarding a digest, you typically want to match the company that produced the enzyme you’re using to the same company for the buffer. If they’re not the same, look up components and concentrations.

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#

03/28/13

This looks to be the same size no matter the temperature. We hypothesize that either our primers are laying down non-specifically or they are oriented in the wrong direction. If it turns out our primers are fine, then we may have a different organis, than expected.

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#

03/27/13

**PCR repeat of Clostridial origin repeat**

Preformed as outline on page 15 with the following modifications

+ Only a 1:100 dilution was used since our DNA template had a concentration of 164.6 ng/μL, which is a good value to use for genomic DNA.

* A temperature gradient for the annealing step was setup where different columns in thermocycler are a different temperature during the annealing process.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Column | Temperature (μC) || 2 | 50.2μC || 3 | 50.8μC || 4 | 51.7μC || 5 | 52.8μC || 6 | 54.1μC || 7 | 55.4μC || 8 | 56.7μC || 9 | 57.9μC || 10 | 58.8μC || 11 | 59.5μC ||

To set up temperature gradient on thermocycler

1. Highlight stage of interest (in this case; annealing)
2. Selection "options"
3. Select "Show Gradient"

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#

03/25/13

**Gel of Clostridial Origin PCR Product**

PCR analysis was completed March 13, 2013

* well 1:Ladder
* well 2:10 Dilution
* well 3:100 Dilution
* well 4:1000 Dilution
* well 5:10000 Dilution
* well 6:Control

From these gels, we can see a band between 250 and 500 bases, which isn't the size of our clostridial origin. Assuming PCR worked correctly, we should see a band of approximately 1200 base pairs. Since we ran multiple cells with the same result, we are hypothesizing that an error was in the PCR reaction. Therefore, we are going to repeat PCR before we move forward by adjusting the annealing temperature.

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#

03/15/13

**Quantification of PCR Product and Linearized Vector with Chloramphenicol**

Nanodrop Protocol

1. Click icon with ND-1000 on computer
2. Click "Nucleic Acids"
3. Wipe off pedestal with chemwipe
4. Load 3°L DI water to initialize and click blank
5. Click "Measure" to verify flat line
6. Load 3°L of sample
7. Click "Measure"
8. Click "Print" screen after sample ID has been typed in

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#

03/13/13

Making and Preparing Agarose Gel

1. In a 125mL erlenmyer flask, combine 0.4g ultra pure agarose and 40mL 1x TAE buffer, which makes a 1% gel
2. Swirl to mix. Place in microwave for 40 seconds. If all agarose isn't dissolved, heat again in 7 second increments
3. Run flask bottom under water to cool agarose
4. Pour into gel rig with comb inserted
5. Let gel cool until opaque

Running Gel of Post-Digested pSB1C3 and pSB1A3

1. Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
2. Remove comb slowly
3. Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL- - Load samples into wells after 1 kb DNA ladder is loaded into far left lane
     * well 1:Ladder
     * well 2:pSB1C3 EcoR1
     * well 3:pSB1C3 EcoR1 Pst1
     * well 4:pSB1C3 Pst1
     * well 5:pSB1A3 EcoR1
     * well 6:pSB1A3 EcoR1 Pst1
     * well 7:pSB1A3 Pst1
     * well 8:pSB1C3 UNDIGESTED- Run gel for 45 minutes-1.5 hours at 85 volts

- Run gel for 45 minutes-1.5 hours at 85 volts

  PCR Reaction Protocol

  1. Combine the following

* 150μL 2x master mix (polymerase,buffer)
* 1μL forward primer
* 1μL reverse primer
* 148μL DI water (PCR water, UV prior to use)

- Make 1:10, 1:100, 1:1000, 1:10000 dilutions of template
- In four tubes, combine 50μL of step 1 solution and 1μL of diluted template
- in fifth tube, only put in step 1 solution as a negative control

Regular PCR Cycle

1. 95°C for 45 seconds
2. 55°C for 1 minute
3. 75°C for 1.5 minutes
4. These three steps are cycled 35 times

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#

03/12/13

Preformed the following restriction digest of pSB1A3 and pSB1C3.

500ng DNA in 20μL

pSB1A3 [DNA] = 135.8ng/μL

pSB1C3 [DNA] = 74.7ng/μL

500ng x μL/135.8μg = 3.68μL

500ng x μL/74.7μg = 6.69μL

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | EcoRI | PstI | 10X Buffer | DNA | PCR Water | TOTAL || pSB1A3 | 2μL | 0μL | 2μL | 4μL | 12μL | 20μL || pSB1A3 | 2μL | 2μL | 2μL | 4μL | 10μL | 20μL || pSB1C3 | 2μL | 0μL | 2μL | 7μL | 9μL | 20μL || pSB1C3 | 2μL | 2μL | 2μL | 7μL | 7μL | 20μL ||

![Smiley face](https://static.igem.org/mediawiki/2013/b/b3/12.1.png)

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#

03/8/13

**Nanodrop DNA Quanitification**

Protocol

1. Open Nanodrop 7000 V.6.0
2. Select Nucleic Acid
3. Blank via 3μL of PCR water
4. Verify 0ng/&#;L DNA in PCR water
5. Load 3μL of sample
6. Record DNA concentration in ng/μL
7. Print Screen

pSB1K3

![](https://static.igem.org/mediawiki/2013/e/e7/K3.png)