iGEM 2015 — Team Missouri_Rolla

# Team:Missouri Rolla/Notebook

[![](https://static.igem.org/mediawiki/2015/1/16/Mstigem_logo2015.png)](https://2015.igem.org/Team%3AMissouri_Rolla)
![Missouri cave photographed by Lynn Dieter](https://static.igem.org/mediawiki/2015/d/d1/Mstigem_cave_background.jpg)

[Project](https://2015.igem.org/Team%3AMissouri_Rolla)[Human Practices](https://2015.igem.org/Team%3AMissouri_Rolla/Practices)
[Parts](https://2015.igem.org/Team%3AMissouri_Rolla/Parts)
[Notebook](https://2015.igem.org/Team%3AMissouri_Rolla/Notebook)[Team](https://2015.igem.org/Team%3AMissouri_Rolla/Team)
[Safety](https://2015.igem.org/Team%3AMissouri_Rolla/Safety)[Attributions](https://2015.igem.org/Team%3AMissouri_Rolla/Attributions)

# NOTEBOOK

Many of our standard procedures can be found in our [Lab Training Manual](https://static.igem.org/mediawiki/2015/0/09/Mstigem_ltm_e2.pdf).

**Date: 5/22/2015
People in lab: Kent Gorday**
**Electrical Transformation of 2015 iGEM Plate 1 3O**
**Start Time:** 3:00pm
**Purpose:** To transform and clone a promoter and rbs in standard iGEM backbone for later addition of last year's hmp part.
**Protocol:** 1.5µL of BBa\_K608002, previously taken from iGEM Plate 1 3O, was electrically transformed into competent cell. An 1.8kV pulse was applied according to Lab Manual procedure. Plates were left to incubate overnight.
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 5/22 ET1 KG 20µL | BBa\_K608002 in E. coli on chloramphenicol, 20 µL plating volume | 2015 iGEM Plate 1 3O |
| 5/22 ET1 KG 200µL | BBa\_K608002 in E. coli on chloramphenicol, 200 µL plating volume | 2015 iGEM Plate 1 3O |

**Stop Time:** 4:10pm
**Next:** Inoculate liquid culture from 5/22 ET1 then miniprep product and glycerol stock of hmp.

**Date: 5/25/2015
People in lab: Kira Buckowing**
**InocµLations of hmp frozen stock and colonies from ET1 5/22 200µL plate**
**Start Time:** 3:00pm
**Purpose:** To have liquid culture grown up of hmp and the iGEM kit parts (Promoter + rbs)
**Protocol:** Inoculation of 2 broth cultures from each the hmp frozen stock and the ET1 5/22 200µL plate. Wire loop was used for 3 of the 4 total samples, pipette for the last hmp broth culture. Procedure taken from iGEM LTP Lab Manual edition 2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| BC1 KKB 5/25 | iGEM kit part, liquid culture | ET1 5/22 | 2 |
| BC3 KKB 5/25 | hmp stock liquid culture | hmp glycerol stock | 2 |

**Stop Time:** 3:25pm
**Next:** Minipreps of all samples to isolate the desired plasmids.

**Date: 5/26/2015
People in lab: Kent Gorday**
**Miniprep and digestion of hmp, pro/rbs, and amp BB**
**Start Time:** 3:09pm
**Purpose:** To extract and digest BBa\_K608002, hmp, and linearized pSB1A3
**Protocol:** Solution miniprep procedure attached to front of lab training manual was used on 3mL of liquid culture. Minipreps 1-4 were analyzed by nanodrop and showed no DNA due to a mistake in miniprep procedure. Identical minipreps were performed using the remaining 2mL of culture. Nanodrop then showed DNA presence, so the following digests were prepared: D1- 20.5 µL pSB1A3, 2.5 µL 10x Tango, 1 µL EcoRI, 1 µL PstI; D2 - 14.5 µL water, 2.5 µL 10x Tango, 6 µL MP5, 1 µL EcoRI, 1 µL SpeI; D3 - 18.5 µL water, 2.5 µL 10x Tango, 2 µL MP7, 1 µL XbaI, 1 µL PstI
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| KG 5/26 MP1, MP2, MP5, MP6 | pro/rbs BBa\_K68002 | 5/25 BC1, BC2 | 4 |
| KG 5/26 MP3, MP4, MP7, MP8 | hmp | 5/25 BC1, BC2 | 4 |
| KG 5/26 D1 | E&P amp BB | Linear pSB1A3 2015 | 1 |
| KG 5/26 D2 | E&S pro/rbs | 5/26 MP5 | 1 |
| KG 5/26 D3 | X&P hmp | 5/26 MP7 | 1 |

**Results:** MP1 - [DNA]: -4.9, 260/280: 1.65, 260/230: 0.06; MP2 - [DNA]: -2.2, 260/280: 1.28, 260/230: 0.04; MP3 - [DNA]: 13.8, 260/280: 1.38, 260/230: 0.35; MP4 - [DNA]: -4.8, 260/280: 1.65, 260/230: 0.08; MP5 - [DNA]: 161.8, 260/280: 1.91, 260/230: 2.51; MP6 - [DNA]: 150, 260/280: 1.87, 260/230: 2.28; MP7 - [DNA]: 508.5, 260/280: 1.85, 260/230: 1.84; MP8 - [DNA]: 462.9, 260/280: 1.82, 260/230: 1.55
**Stop Time:** 7:50pm
**Next:** Ligate 5/26 D1, D2, D3

**Date: 5/27/2015
People in lab: Kira Buckowing**
**Ligation of hmp/pro/rbs/amp BB**
**Start Time:** 3:15pm
**Purpose:** To combine the wanted digest parts from 5/26 into one plasmid (3A assembly)
**Protocol:** Ligation protocol from LM ed2 with the following adjustments: 1µL each of the hmp section and pro/rbs section used for L1 & 2µL each used for L2 with 4µL of the amp backbone
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| L1 KKB 5/27 | 1µL D2, 1 µL D3, 6µL D1; ligated plasmid | 5/26 KG D1, D2, D3 |
| L2 KKB 5/27 | 2µL D2, 1 µL D3, 6µL D1; ligated plasmid | 5/26 KG D1, D2, D3 |

**Stop Time:** 4:25PM
**Next:** Test plasmid with gel electrophoresis or go straight to transformation to see if the plasmid ligated as planned

**Date: 5/28/2015
People in lab: Kent Gorday**
**Electrical transformation of pro/rbs+hmp+amp**
**Start Time:** 3:00pm
**Purpose:** To transform functioning hmp plasmid into competent cells for cloning
**Protocol:** 2µL of the ligations from 5/27 were transformed into electrocompetent cells using a 1.8kV pµLse and LM procedure. Plates were left to incubate overnight.
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 5/28 ET1 | hmp+pro/rbs on amp plate, no arc | 5/27 L1 | 2 |
| 5/28 ET2 | hmp+pro/rbs on amp plate, arced | 5/27 L1 | 2 |
| 5/28 ET3 | hmp+pro/rbs on amp plate, no arc | 5/27 L2 | 2 |
| 5/28 ET3 | hmp+pro/rbs on amp plate, arced | 5/27 L2 | 2 |

**Stop Time:** 4:15pm
**Next:** Inoculate liquid cultures from transformations then miniprep

**Date: 6/7/2015
People in lab: Kent Gorday**
**Inoculation of liquid cultures amp+hmp+pro/rbs**
**Start Time:** 4:25pm
**Purpose:** To clone hmp+pro/rbs/amp plasmid for later miniprep
**Protocol:** According to inoculation protocol, 5µL of ampicillin was added to each 5µL broth culture, then left to incubate overnight
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 6/7 KG BC1 | 1x amp, amp/pro/rbs | 5/28 ET1 20µL | 4 |

**Stop Time:** 4:55pm
**Next:** Miniprep cultures and digest

**Date: 6/8/2015
People in lab: Kent Gorday**
**Observation**
**Start Time:** 2:20pm
**Purpose:** Observe broth cultures
**Notes:** Cultures from 6/7 show only slight growth and were left to incubate again overnight. Preparation of miniprep solutions was completed and solutions put in refrigerator,
**Stop Time:** 3:35pm
**Next:** Miniprep cultures and digest

**Date: 6/9/2015
People in lab: Kent Gorday**
**Inoculation of liquid cultures**
**Start Time:** 11:25am
**Purpose:** To clone hmp/pro/rbs+amp for later miniprep
**Protocol:** 5µL of amp was added to each 5mL LB tube, then a sterile pipette tip used to inoculate the cultures near a flame. Cultures were left in shaking incubator.
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| KG 6/9 BC1 | 1x amp, hmp+pro+rbs | KG 5/28 ET2 20µL | 2 |

**Stop Time:** 11:40am
**Next:** Miniprep cultures and digest

**Date: 6/10/2015
People in lab: Kent Gorday**
**Miniprep, digest, ligation of hmp/pro/rbs**
**Start Time:** 12:50pm
**Purpose:** To produce a completed pro/rbs/hmp part for submission
**Protocol:** Three solution minipreps were performed using 1.5mL of 6/7 BC3 and BC4, and 3mL of 6/9BC1. The amounts of solutions 1,2,3 used per miniprep were 150, 200, and 150µL, respectively. DNA was resuspended in 30µL of TE buffer. Digests: D1 - 10.5µL milliQ water, 2.5µL Tango, 1µL EcoRI, 1 µL PstI, 10µL 6/10MP1; D2 - 3.5µL MilliQ, 2.5µL Tango, 1 µL EcoRI, 1 µL PstI, 17µL 6/10 MP3; D3 - 2.5µL Tango, 1µL EcoRI, 1µL PstI, 20.5 µL pSB1C3 2015. Ligations: L1 - 6µL D3, 2µL D1,1µL 10x T4 buffer, 1µL T4 ligase; L2 - 6µL D3, 2µL D2, 1µL 10x buffer, 1µL T4 ligase; L3 - 4 µL D3, 4µL D1, 1µL 10x buffer, 1µL T4 ligase; L4 - 4µL D3, 4µL D2, 1µL 10x buffer, 1µL T4 ligase
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| KG 6/10 MP1,2 | hmp+pro/rbs+amp | 6/7 BC3, 4 | 2 |
| KG 6/10 MP3 | hmp+pro/rbs+amp | 6/9 BC1 | 1 |
| 6/10 KG D1, 2 | pro/rbs+hmp cut E&P | 6/10 KG MP1, 3 | 2 |
| 6/10 D3 KG | chlorBB cut E&P | pSB1C3 2015 | 1 |
| 6/10 KG L1, 2, 3, 4 | completed hmp+pro/rbs part for igem | 6/10 D3&D1, D2&D3, D3&D1, D3&D2 | 4 |

**Results:** MP1 - [DNA]: 100, 260/280: 1.82, 260/230: 1.84; MP2 - [DNA]: 98.5, 260/280: 1.81, 260/230: 1.93; MP3 - [DNA]: 60.7, 260/280: 1.84; 260/230: 2.35
**Stop Time:** 5:00pm
**Next:** Transform ligations to clone and miniprep

**Date: 6/11/2015
People in lab: Kira Buckowing, Kent Gorday**
**Electrical transformation of hmp/rbs part and 2J from kit**
**Start Time:** 4:40pm
**Purpose:** To grow colonies with the wanted plasmids
**Protocol:** 2J DNA resuspended in 10µL milliQ, ETs per lab manual
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 2J KKB 6/11/15 | Part BBa\_J04450 in pSB4A5 backbone, rfp in amp | iGEM DNA Plate 4 well 2J | 1 |
| 6F KKB 6/11/15 | Part BBa\_J04450 in pSB3K3 bb, rfp in kan | iGEM DNA plate 4 well 6F | 1 |
| KG 6/11 ET1, ET2, ET3, ET4 | ET of L1, L1, L2, L2, L3 L3 | KG 6/10 L1, L1, L2, L2, L3, L3 | 8 |
| ET5 KKB 6/11 | 2J Kit part ET | ET5 of 2J | 2 |
| ET6 KKB 6/11 | 6F Kit part ET | ET6 of 6F | 2 |

**Notes:** ET6 arced. No growth -KKB 6/13
**Stop Time:** 6:20pm
**Next:** Check for growth

**Date: 6/16/2015
People in lab: Kent Gorday**
**New electrical transformations of hmp/pro/rbs and 2J, 6F kit parts**
**Start Time:** 4:56 pm
**Purpose:** To transform parts fro cloning
**Protocol:** LM ed. 2, D1 through D4 - 17.5µL milliQ, 2.5 Tango, 1µL PstI, and 4µL of 6/10 L1 through L4, respectively.
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 6/16 D1, 2, 3, 4 | hmp/pro/rbs digested with P | KG 6/10 L1, L2, L3, L4 | 4 |
| 6/16 ET1, 2 | hmp/pro/rbs on chlor, plated in duplicates 20 and 200µL | KG 6/10 L1, L2 | 4 |
| 6/16 ET3 | J04450 on amp, plated in duplicates 20/200µL | KKB 6/11 2J | 2 |
| 6/16 ET4 | J04450 on kan, plated in duplicates 20/200µL | KKB 6/11 6F | 2 |

**Stop Time:** 6:35pm
**Next:** If hmp plates grow, prepare part for iGEM submission. Otherwise run gel electrophoresis of digests to diagnose.

**Date: 6/17/2015
People in lab: Kent Gorday**
**Gel electrophoresis of hmp/pro/rbs digested with PstI**
**Start Time:** 4:50pm
**Purpose:** To troubleshoot hmp/pro/rbs using gel electrophoresis
**Protocol:** LM ed2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 3 | 5µL low range ladder |
| 4 | 6/16 D1 15µL |
| 5 | 6/16 D2 15µL |
| 6 | 6/16 D3 15µL |
| 7 | 6/16 D4 15µL |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/c/cd/Mstigem_jun17.jpeg)
**Notes:** Calculated fragment lengths for all ligations: 2300bp, 4000bp
**Stop Time:** 6:48pm
**Next:** Run gel of MP 5/26 and digests from 6/10

**Date: 6/18/2015
People in lab: Kent Gorday**
**Gel electrophoresis of 6/10 digests and 5/26 MP**
**Start Time:** 7:20pm
**Purpose:** To continue troubleshooting hmp/pro/rbs using gel electrophoresis
**Protocol:** LM ed2, Digests: D1 - 11.5 MilliQ, 2.5µL Tango, 10µL 5/26 P1, 1µL PstI
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 2 | 5µL low range ladder |
| 3 | 6/16 D1, 10µL |
| 4 | 6/18 D1, 15µL |
| 5 | 6/10 D1, 10µL |
| 6 | 6/10 D3 15µL |

**Stop Time:** 11:00pm
**Next:** Repeat transformation of 5/27 L1 & 2 with new ampicillin plates

**Date: 6/19/2015
People in lab: Kent Gorday**
**Gel purification and ligation of hmp/pro/rbs**
**Start Time:** 6:00pm
**Purpose:** To assemble pro/rbs/hmp using gel purification
**Protocol:** LM ed2, Digest D1 - 20.5 µL 2015 pSB1C3, 2.5 µL Tango, 1µL EcoRI, 1µL PstI, Ligation: L1 - 4µL 6/19 GE1, 2µL GE2, 2µL GE3, 1µL buffer, 1µL T4 ligase
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 1 | 6/19 D1, 15µL |
| 2 | 5/26 D2, 15µL |
| 3 | 5/26 D3, 15µL |
| 4 | Low range ladder 5µL |

**Results:** Nanodrop showed no DNA in gel purifications.
**Stop Time:** 10:45pm
**Next:** Repeat transformation of 5/27 L1 & 2 on new ampicillin plates

**Date: 6/20/2015
People in lab: Kent Gorday**
**Electrical transformations of hmp+pro/rbs on amp/chlor**
**Start Time:** 2:52pm
**Purpose:** to clone hmp+pro/rbs plasmids
**Protocol:** 1µL of ligations transformed into ecomp cells
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 6/20 ET1 | hmp/pro/rbs on amp | 5/27 L1 | 2 |
| 6/20 ET2 | hmp/pro/rbs on amp | 5/27 L2 | 2 |
| 6/20 ET3 | hmp/pro/rbs on chlor | 6/20 L1 | 2 |

**Stop Time:** 4:00pm
**Next:** Inoculate liquid cultures and miniprep

**Date: 6/22/2015
People in lab: Kent Gorday**
**Inoculation of liquid cultures of hmp/pro/rbs/amp**
**Start Time:** 9:52am
**Purpose:** To clone plasmid for later miniprep
**Protocol:** 5µL amp added to tubes, inoculated with sterile pipette tips
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 6/22 BC1 |  | 6/20 ET2 | 2 |

**Stop Time:** 10:10am
**Next:** Miniprep and possibly find different amp plates

**Date: 6/22/2015
People in lab: Kira Buckowing**
**ET of 2J and hmp/amp**
**Start Time:** 4:35 pm
**Purpose:** To transform wanted plasmids into ecoli for later use
**Protocol:** LM ed2
**Exceptions:** 2µL each of hmp used
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| ET1 6/22 KKB | 2J kit parts, RFP | 2J KKB | 2 |
| ET2 6/22 KKB | hmp/pro/rbs in amp | hmp L1 5/27 | 2 |
| ET3 6/22 KKB | hmp/pro/rbs in amp | hmp L2 5/27 | 2 |

**Notes:** ET3 popped, ET2 was spilled
**Stop Time:** 6:35pm
**Next:** Inoculation of broth cultures

**Date: 6/23/2015
People in lab: Kira Buckowing**
**Inoculation of pro/rbs/hmp, 2J, 6F kit parts**
**Start Time:** 4:45pm
**Purpose:** To obtain liquid cultures for minipreps
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Souce Label | Quantity |
| ET3 BC1 KKB |  | ET3 6/22 | 1 |
| ET BC2 KKB | 1 | ET2 6/22 | 1 |
| ET1C BC3, 4 KKB |  | colony from lid of dish 2J | 2 |
| BC5, 6 KKB |  | colony of 6F | 2 |

**Stop Time:** 5pm
**Next:** miniprep

**Date: 6/24/2015
People in lab: Kent Gorday**
**Start Time:** 7:55pm
**Purpose:** To assemble pro/rbs/hmp/chlor and ensure identity of product
**Protocol:** Kitless minipreps procedure. Digests: D1 - 18.5 milliq, 2.5 tango, 2 6/24 MP1, 1 EcoRI, 1 PstI; D2 - 10.5 milliq, 2.5 tango, 10 6/24 MP3, 1µL EcoRI, 1µL PstI; D3 - 10.5milliq, 2.5 tango, 10 pSB1C3 2014, 1µL EcoRI, 1µL PstI. Ligation: L1 - 5µL D3, 3µL D2, 1µL buffer, 1µL ligase
**Exceptions:** 150µL of soln 1, 200 µL of soln 2, 175µL of sln 3, 550µL isopropyl alcohol.
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Souce Label | Quantity |
| 6/24 MP1 | 2J plate 4, lid colony | 6/23 BC4ET1C | 1 |
| 6/24 MP2, 3 | hmp/pro/rbs amp | 6/22 BC1, 2 | 2 |
| 6/24 ET1, 2 | hmp/pro/rbs/amp | 5/27 L1,2 | 4 |
| 6/24 D1 | 2J digested E&P | 6/24 MP1 | 1 |
| 6/24 D2 | hmp/pro/rbs digested E&P | 6/24 MP3 | 1 |

**Results:** MP1 - [DNA]: 524.4ng/µL, 260/280: 1.85, 260/230: 2.32; MP3 - [DNA]: 96/73, 260/280: 1.82, 260/230: 1.75
**Notes:** 6/24 D3,pSB1C3 2014, linear B digested E&P; 6/24 L1, 6/24 D2&D3, hmp/pro/rbs chlor
**Stop Time:** 12:01am
**Next:** Run gel of digests and transform ligations if lengths are correct.

**Date: 6/25/2015
People in lab: Kent Gorday**
**Gel elecetrophoresis of MPs**
**Start Time:** 4:30pm
**Purpose:** to ensure identity of miniprep products
**Protocol:** LM ed2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 3 | 5µL low range ladder |
| 4 | 10µL 6/24 D1 |
| 5 | 10µL 6/24 D2 |

**Stop Time:** 5:40pm
**Next:** religate hmp/pro/rbs amp

**Date: 6/27/2015
People in lab: Kent Gorday**
**New ligation of hmp/pro/rbs+amp**
**Start Time:** 8:55pm
**Purpose:** To porform new ligations of 5/26 D1, 2, and 3 since gel electrophoresis showed failed of 5/27 ligations
**Protocol:** the following ligations were prepared and left to react overnight, L1 - 5/26 D1 7µL, 0.8µL D3, 0.2µL D2, 1µL 10x buffer, 1µL T4 ligase
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 6/27 L1 | hmp/pro/rbs + amp | 5/26 D1, D2, D3 |

**Stop Time:** 9:35 pm
**Next:** Transform ligation, culture, and miniprep

**Date: 6/28/2015
People in lab: Kent Gorday**
**Electrical transformation of kit parts and hmp/pro/rbs/amp**
**Start Time:** 6:05pm
**Purpose:** to clone plasmids for later inoculation and miniprep
**Protocol:** 10µL MilliQ water was used to resuspend 2014 plate 4 6F. Four electrical transformations were prepared according to the lab manual.
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 6/28 ET1, 2 | hmp/pro/rbs/amp | 2µL 6/27 L1 | 2 |
| 6/28 ET3 | 2J on amp | 1µL 2015-4-2J | 1 |
| 6/28 ET4 | 6F on kan | 1µL 2014-4-6F | 1 |

**Stop Time:** 7:30pm
**Next:** Inoculate liquid cultures

**Date: 6/30/2015
People in lab: Kira Buckowing**
**gBlock DNA resuspension and restriction digest**
**Start Time:** 4:40pm
**Purpose:** To get gBlock DNA into a usable form to prep the pSB1C3 backbone for project work.
**Protocol:** Digest as per Lab Manual and resuspensions as per IDT instructions: 1. Centrifuge tube for a few seconds 2. add TE up to 10 ng/µL 3. Vortex briefly 4. Incubate @ 50C for 20 min 5. Vortex and centrifuge again briefly 6. [Added step] transfer total to pcr tube
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| pSB1C3 Digested 6/30 | pSB1C3 cut with E&P | pSB1C3 kit DNA | 1 |
| 6/30/15 KKB 1-1, 1-2, 1-3, 1-4 | 10ng/µL DNA | Part 1 1of4, 2of4, 3of4, 4of4 | 4 |
| 6/30/15 KKB, 2.1-1,2.1-2,2.1-3,2.1-4 | 10ng/µL DNA | Part 2-1 1of4, 2of4, 3of4, 4of4 | 4 |
| 6/30/15 KKB, 2.2-1,2.2-2,2.2-3,2.2-4 | 10ng/µL DNA | Part 2-2 1of4, 2of4, 3of4, 4of4 | 4 |
| Part3 6/30/15 KKB | 10ng/µL DNA | Part 3 ocimene synthase | 1 |

**Notes:** 100µL added to 2.2-1 instead of the calculated 50µL so the concentration is halfed from the expected. 2014 linearized pSB1C3 used.
**Stop Time:** 7:00pm
**Next:** Assembly of the gBlock DNA fragments

**Date: 6/30/2015
People in lab: Kent Gorday**
**Electrical transformation of Kit parts and hmp/pro/rbs+amp**
**Start Time:** 8:30pm
**Purpose:** to clone plasmids for later inoculation and miniprep
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| KG 6/30 ET1 | hmp/pro/rbs on amp | 6/27 L1 | 2 |
| KG 6/30 ET2 | pSB4A5 on amp | 2015 plate4 2J | 2 |
|KG 6/30 ET3 pSB3K3 on kan | 2014 plate4 6F | 1 | 2 |

**Stop Time:** 9:40pm
**Next:** inoculate broth cultures and miniprep

**Date: 7/2/2015
People in lab: Kent Gorday**
**Chemical transformations ond PCR of hmp for gst tagging**
**Start Time:** 8:27pm
**Purpose:** clone plasmids and prepare hmp for gst tagging
**Protocol:** LM ed2, PCR - A1: 12.5 µL Taq MM, 11.3µL MilliQ, 0.2µL 5/26 MP7, 0.5 µL F hmp, 0.5 µL R hmp, Ligation: L1 - 1µL 10x buffer, 5.1 µL 5/26 D1, 2.4 µL 5/26 D2, 0.5 µL 5/26 D3, 1µL ligase
**Exceptions:** PCR Program: 94C - 2mins, 32 cycles of (94C - 26s, 55C- 40s, 68C - 3min), 68C - 7min, 4C - hold
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/2 BC1 | 2014 6F in kan | 6/30 ET3 | 2 |
| 7/2 BC3 | hmp in pSB1C3 | hmp glycerol stock | 1 |
| 7/2 A1 | hmp prepaged for gst tagging | 5/26 MP7 | 1 |
| 7/2 CT1 | hmp/pro/rbs/amp | 6/27 L1 | 1 |
| 2015 plate 4 well 2J | 2015 2J plate4 | 1 | 1 |

**Notes:** 7/2 L1, 5/26 D1. D2, D3, hmp/pro/rbs/amp
**Stop Time:** 1:11am
**Next:** Run a gel of A1, 7/2 L1, 6/27 L1, miniprep BCs incolµLate new BCs from Cts

**Date: 7/3/2015
People in lab: Kent Gorday**
**Miniprep hmp and pSB3K3**
**Start Time:** 3:30pm
**Purpose:** Obtain plasmids from culture
**Protocol:** Three minipreps were performed using the kitless miniprep protocol
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 7/3 MP1 | pSB3K3 in TE | 7/2 BC1 |
| 7/3 MP2 | pSB3K3 in TE | 7/2 BC2 |
| 7/3 MP3 | hmp in pSB1C3 | 7/2 BC3 |

**Results:** MP2 - [DNA]: 320.1 ng/µL, 260/280: 1.89, 260/230: 2.38; MP3 - 216.4, 1.75, 1.8
**Stop Time:** 5:00pm
**Next:** ET of hmp/pro/rbs

**Date: 7/3/2015
People in lab: Kent Gorday**
**Electrical transformation of hmp/pro/rbs/amp**
**Start Time:** 5:10pm
**Purpose:** assemble hmp in pSB1C3
**Protocol:** LM Ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/3 ET1 | hmp/pro/rbs amp | 7/2 L1 | 2 |

**Stop Time:** 6:22pm
**Next:** If no growth, run a new digestion of pSB1A3 and repeat 7/2 L1 using new BB. Inoculate BC and MPs from 7/2 CT2, then digest pSB3K3 and pSB4A5 to check by gel

**Date: 7/4/2015
People in lab: Kent Gorday**
**Inoculation of liquid cultures for pSB4A5**
**Start Time:** 10:14 am
**Purpose:** clone pSB4A5 for later miniprep
**Protocol:** Two broth cultures were inoculated using a wire loop
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/4 BC1 | RFP in pSB4A5 | 7/2 CT2 | 2 |

**Stop Time:** 10:22am
**Next:** Miniprep broth cultures

**Date: 7/5/2015
People in lab: Kent Gorday**
**Miniprep and digestion of pSB4A5, pSB3K3, pSB1A3**
**Start Time:** 7:35pm
**Purpose:** prepare pSB4A5 and pSB3K3 for gel and use
**Protocol:** Kitless miniprep protocol and LM ed2, Digests: D1 - 1µL E, 1µL P, 2.5µL tango, 11.5 water, 9µL 7/3 MP2; D2 - 1 E, 1 P, 2.5 buffer, 20.5 7/5 MP2; D3 - 1 E, 1 P, 2.5 buffer, 20.4 pSB1A3
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/5 MP1 | RFP in pSB4A5 | 7/4 BC1 | 1 |
| 7/5 MP2 | RFP in pSB4A5 | 7/4 BC2 | 1 |
| 7/5 D1, D2 | RFP in pSB3K3 cut E&P | 7/3 MP2 | 2 |
| 7/5 D3 | pSB1A3 cut E&P | 2015 Linear pSB1A3 | 1 |

**Results:** MP1 - [DNA]: 69.37, 260/280: 1.87, 260/230: 1.92; MP2 - 138.52, 1.71, 1.46
**Stop Time:** 10:30pm
**Next:** Run gel of digests, repeat 7/2 L1 using 7/5 D3

**Date: 7/6/2015
People in lab: Kent Gorday**
**Ligation of pro/rbs/hmp amp**
**Start Time:** 9:51am
**Purpose:** assemble hmp/pro/rbs part
**Protocol:** L1 - 1µL ligase, 1µL buffer, 1.4 µL D3, 5µL D2, 1.6µL D3
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 7/6 L1 | hmp/pro/rbs/amp | 7/5 D3, 5/26 D2, 5/26 D3 |

**Stop Time:** 10:10am
**Next:** Run gel of 7/5 digests, transform ligation

**Date: 7/6/2015
People in lab: Kira Buckowing**
**HiFi assembly Part 1**
**Start Time:** 10:15am
**Purpose:** To assemble part 1
**Protocol:** Following mixed in PCR tube and heated to 50C for 1 hr, HiFi - HiFi Part 1: 1.3µL pSB1C3, 1-1 2.6µL, 1-2 3.1µL, 1-3 3.1µL, 1-4 3.6µL, HiFi MM 13.2
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| HiFi part 1 7/6 KKB | Assembled Part 1 for project | pSB1C3 6/30, 1-1, 1-2, 1-3, 1-4 6/30 |

**Notes:** Slightly less than an hour incubation
**Stop Time:** 11:25pm
**Next:** Tramsform and gel/digest to check product

**Date: 7/6/2015
People in lab: Kent Gorday**
**Gel of backbone digests**
**Start Time:** 11:28am
**Purpose:** To check identity of digests
**Protocol:** LM ed2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 3 | Ladder 5µL |
| 4 | 7/5 D1 10µL |
| 5 | 7/5 D2 10µL |
| 6 | 7/5 D3 10µL |

**Stop Time:** 12:55pm
**Next:** Transform 7/6 L1

**Date: 7/6/2015
People in lab: Kira Buckowing**
**HiFi Assembly Part 2-1**
**Start Time:** 1pm
**Purpose:** To assemble the Part 2-1
**Protocol:** The following were mixed in a PCR tube and incubated @ 50C for an hour: pSB1C3 1.3µL, 2.1-1 4.6µL, 2.1-2 2.8µL, 2.1-3 2.8µL, 2.1-4 3.1µL, HiFi MM 14.6 µL
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| HiFi Part 2.1 7/6 KKB | Part 2-1 for project | pSB1C3 digested 6/30, 2.1-1, 2.1-2, 2.1-3, 2.1-4 6/30 |

**Stop Time:** 2:20pm
**Next:** Transformation into e. coli to check products

**Date: 7/6/2015
People in lab: Kira Buckowing**
**Assembly of part 2-2**
**Start Time:** 4:30pm
**Purpose:** To assemble the 2-2 part
**Protocol:** The following were mixed in a PCR tube and incubated at 50C for an hour. HiFi assembly: Part 2.2 - pSB1C3 1.3µL, 2.2-1 2.8µL, 2.2-2 2.8µL, 2.2-3 2.8µL, 2.2-4 3.1µL, HiFi MM 12.8µL
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| HiFi Part 2.2 7/6 KKB | Part 2-2 for project work | pSB1C3 6/30 2.2-1, 2, 3,4 6/30 |

**Stop Time:** 5:50pm
**Next:** Transformation and gel to check for product correctness

**Date: 7/6/2015
People in lab: Kent Gorday**
**CT of HiFi assemblies and hmp/pro/rbs**
**Start Time:** 7:10pm
**Purpose:** Transform HiFi assemblies to clone and verify plasmids and assembled hmp/pro/rbs part
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/6 BC1, 2, 3 | FP pSB4A5 | 7/2 CT2 | 3 |
| 7/6 ET1 | hmp/pro/rbs | 7/6 L1 | 2 |
| 7/6 ET2 | part 1 on chlor | HiFi part 1 7/6 | 2 |
| 7/6 ET3 | part 2-1 on chlor | HiFi part 2-1 7/6 | 2 |
| 7/6 ET4 | Part 2-2 on chlor | HiFi part 2-2 7/6 | 2 |

**Stop Time:** 10:00 pm
**Next:** pSB4A5 and inoculte BC from transformations

**Date: 7/7/2015
People in lab: Kira Buckowing**
**Digest and gel of HiFi assemblies**
**Start Time:** 5:15pm
**Purpose:** To check validity of products
**Protocol:** LM ed 2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 1 | Ladder |
| 2 | D1 - Part 1 |
| 3 | D2 - Part 2-1 |
| 4 | D3 - Part 2-2 |

**Notes:** New purple ladder and dye used
**Stop Time:** 8:00pm
**Next:** Try again, no band

**Date: 7/7/2015
People in lab: Kent Gorday**
**CT and miniprep of pSB4A5**
**Start Time:** 9:00pm
**Purpose:** obtain pSB4A5 and clone plasmids
**Protocol:** Two minipreps were prepared, but the first was lost. 7/7 MP2 used 3mL of culture. Digests: D1 - 12.5 µL water, 2.5 tango, 8µL MP2, 1µL E, 1µL P
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/7 MP2 | RFP pSB4A5 | 7/6 BC3 | 1 |
| 7/7 D1 | psb4a5 cut e&p | 7/7 MP2 | 1 |
| 7/7 CT1 | hmp/pro/rbs amp | 7/6 L1 | 1 |
| 7/7 CT2, 3, 4 | Part1, 2-1, 2-2 on chlor | 7/6 HiFi part 1, 2-1, 2-2 | 4 |

**Results:** MP2 - [DNA]: 371, 260/280: 1.76, 260/230: 1.36
**Stop Time:** 12:13am
**Next:** Run gel of 7/7 and 7/2 A1, inoculate BCs from CTs

**Date: 7/8/2015
People in lab: Kent Gorday**
**Inoculation of BCs**
**Start Time:** 9:30am
**Purpose:** clone hmp/pro/rbs amp
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/8 BC1, 2 | hmp/pro/rbs amp | 7/7 CT1 | 2 |
| 7/8 BC3, 4 | hmp/pro/rbs amp | 7/2 CT2 | 2 |

**Stop Time:** 9:50am
**Next:** Miniprep BCs, run gel of 7/7 D1 and 7/2 A1

**Date: 7/9/2015
People in lab: Kent Gorday**
**Minirpep, digest, and gel of pSB4A5, HiFi assemblies and hmp/pro/rbs**
**Start Time:** 4:00pm
**Purpose:** obtain plasmids, verify backbone, parts, and assemblies
**Protocol:** Kitless miniprep procedure, LM ed. 2. Digests: D1 - 14.5µL MP1, 6µL water, 2.5 tango, 1 E, 1 P; D2 - 6µL MP3, 14.5 water, 2.5 tango, 1 E, 1 P. Ligation: 1µL buffer, 3.8 µL 7/9 D1, 4.2 6/30 D1, 1µL ligase
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 1 | Hifi part 1 7/6 5µL |
| 2 | purple ladder, 10µL |
| 3 | 7/2 A1, 10µL |
| 4 | 7/7 D1, 10µL |
| 5 | 7/9 D2, 10 µL |
| 6 | 7/9 D1, 10µL |
| 7 | Hifi part 2-1 7/6 5µL |
| 8 | Hifi part 2-2 7/6 5µL |

**Results:** MP1 - [DNA]: 172.9ng/µL, 260/280: 1.79, 260/230: 1.38; MP2 - 72, 1.81, 1.45; MP3 - 496.9, 1.82, 1.55.
![gel electrophoresis](https://static.igem.org/mediawiki/2015/c/c1/Mstigem_jul9.jpeg)
**Stop Time:** 11:55pm
**Next:** Analyze gel to determine if PCR product shoµLd be purified and ligated and transformed.

**Date: 7/11/2015
People in lab: Kent Gorday**
**tansformation efficiency and hmp/pro/rbs assembly**
**Start Time:** 6:10pm
**Purpose:** Assess transformation efficiency of new electrocomp cells and assemble hmp/pro/rbs
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Lable | Description | Source Label | Quantity |
| 7/11 ET1, 2 | pro/rbs/hmp in chlor | 7/9 L1 | 4 |
| 7/11 ET3 | hmp/pro/rbs in amp | 7/6 L1 | 2 |
| 7/11 ET4, 5 | Trans eff. kit. 2015 10pg/µL | RFP in chlor | 4 |

**Stop Time:** 7:30pm
**Next:** calculate efficiency, Miniprep , obtain GST plasmid, digest it and 7/2 A1

**Date: 7/12/2015
People in lab: Kent Gorday**
**Chemical transformation of hmp/pro/rbs**
**Start Time:** 8:25pm
**Purpose:** to clone hmp/pro/rbs for later use
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/12 CT1, 2 | pro/rbs/hmp in chlor | 7/9 L1 | 4 |
| 7/12 CT3 | pro/hmp/rbs in amp | 7/6 L1 | 2 |

**Notes:** 7/11 ET5 had a single red colony, all other empty
**Stop Time:** 11:40pm
**Next:** make new electrocomp and SOC, incoluate and miniprep growth

**Date: 7/13/2015
People in lab: Kira Buckowing**
**HiFi Assembly**
**Start Time:** 6:45pM
**Purpose:** Correctly assemble plasmids for project work
**Protocol:** HiFi dna assembly protocol (same as last time except correct MM used)
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| HiFi part 1 7/13 | Part 1 | gblocks + pSB1C3 |
| HiFi part 2-1 7/13 | Part 2-1 | gblocks + pSB1C3 |
| HiFi part 2-2 7/13 | Part 2-2 | gblocks + pSB1C3 |

**Stop Time:** 7:15pm
**Next:** Trasformation to check

**Date: 7/13/2015
People in lab: Kent Gorday**
**CT of HiFis**
**Start Time:** 8:00pm
**Purpose:** to clone and screen HiFi assemblies
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/13 CT1 | Part 1 on chlor+glucose | 7/13 HiFi Part1 | 2 |
| 7/13 CT2 | part 2-1 chlor on glucose | 7/13 HiFi Part 2-1 | 2 |
| 7/13 CT3 | part 2-2 chlor on glucose | 7/13 HiFi Part 2-2 | 2 |

**Stop Time:** 11:20pm
**Next:** Inoculate BCs from CTs, miniprep

**Date: 7/14/2015
People in lab: Kent Gorday**
**Restriction digest and gel electrophoresis of HiFi assemblies**
**Start Time:** 3:30pm
**Purpose:** Verify hiFi assembly products and transform
**Protocol:** LM ed2, Digests: D1 - 10µL HiFi part 1, 10.5 µL water, 2.5µL tango, 1 E, 1 P; D2 - 10µL part 2-1, 10.5 water, 2.5 tango, 1 E, 1 P; D3 - 10µL Part 2-2, 10.5 µL water, 2.5 tango, 1µL E, 1µL P
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 2 | Purple, 2-log ladder |
| 3 | 7/14 D1 10µL |
| 4 | 7/14 D2 10µL |
| 5 | 7/14 D3 10µL |
| 6 | 7/6 L1 5µL |
| 7 | 7/9 L1 5µL |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/c/cd/Mstigem_jul14.jpeg)
**Stop Time:** 6:30pm
**Next:** Transform 7/13 HiFi part 1, 2-1, 2-2

**Date: 7/14/2015
People in lab: Kira Buckowing**
**CTs**
**Start Time:** 5pm
**Purpose:** Transform the wanted plasmids into ecoli
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/14 CT1 | HiFi assemblies plasmids in e. coli | HiFi Part 1 | 2 |
| 7/14 CT2 | HiFi assemblies plasmids in e. coli | HiFi Part 2-1 | 2 |
| 7/14 CT3 | HiFi assemblies plasmidsin e. coli | HiFi Part 2-2 | 2 |

**Stop Time:** 7:10pm
**Next:** check for colonies

**Date: 7/16/2015
People in lab: Kira Buckowing**
**Inoculations**
**Start Time:** 9:00pm
**Purpose:** To prepare liquid cultures for minipreps
**Protocol:** LM Ed2
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| BC1 7/16 KKB | Possibly Part1 | 7/14 CT1 |
| BC2 7/16 KKB | Possibly Part1 | 7/14 CT1 |

**Stop Time:** 9:15 pm
**Next:** minipreps

**Date: 7/17/2015
People in lab: Kent Gorday**
**Miniprep, digest and gel of part 1 in psb1c3**
**Start Time:** 3:10pm
**Purpose:** obtain and verify Part1 and pSB1C3
**Protocol:** LM ed2, three glycerol stocks were prepared, three kitless minipreps. Digest: D1 - 20.5µL MP1, 2.5µL 10x tango, 1µL E, 1µL P
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 7/17 MP1 | Possible part 1 in chlor, [DNA]:109.37, 260/280:1.82, 260/230: 1.8 | 7/16 BC1 |
| 7/17 MP2 | Possible Part 1 in chlor, [DNA]: 98.44, 260/280:1.79, 260/230:1.73 | 7/16 BC2 |
| 7/17 MP3 | Part 1 in chlor, [DNA]: 67, 260/280:1.73, 260/230: 1.04 | 7/16 BC2 |
| 7/17 D1 | Possible part 1 cut E&P | 7/17 MP1 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/b/b6/Mstigem_jul17.jpeg)
**Stop Time:** 9:00pm
**Next:** Inoculate new BCs and try minipreps again

**Date: 7/20/2015
People in lab: Kent Gorday**
**Transformation efficient of new electrocomp cells**
**Start Time:** 3:00pm
**Purpose:** To test new electro comp cells and clone part 1
**Protocol:** Two BCs were made, two ETs were performed, LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/20 BC1, 2 | Possible part 1 | 7/16 BC1, 2 | 2 |
| 7/20 ET1, 2 | RFP in chlor | Trans. eff. kit | 4 |

**Stop Time:** 4:40pm
**Next:** calculate transformation efficiency and transform hifi assemblies

**Date: 7/21/2015
People in lab: Kent Gorday**
**Miniprep, digest, Transformation of HiFi assemblies**
**Start Time:** 1:30pm
**Purpose:** transform HiFi assemblies into competent cells
**Protocol:** LM Ed2. Digests: D1 - 2.5µL tango, 1 E, 1 P, 20.5 µL 7/21 MP1
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/21 MP1, 2 | Poss. part1 | 7/20 MP2 | 2 |
| 7/21 D1 | Poss.part1 cut E&P | 7/21 MP1 | 1 |
| 7/21 CT1 | part1 | Part 1 | 1 |
| 7/21 CT2 | part2-1 | Part2-1 | 1 |
| 7/21 CT3 | part2-1 | Part2-2 | 1 |

**Results:** MP1 [DNA]: 50, 260/280: 1.82, 260/230: 1.36; MP2 [DNA]: 17, 1.9, 1.8
**Stop Time:** 9:00pm
**Next:** continue transforming, pcr assembled parts to verify

**Date: 7/22/2015
People in lab: Kent Gorday**
**PCR of HiFi assemblies**
**Start Time:** 2:01am
**Purpose:** verify hiFi assembly
**Protocol:** LM ed2, PCR: A1 - 10µL taq MM, 0.5 VR, 0.5 VF2, 9µL 7/13 hifi part1
**Exceptions:** PCR program: 94C 3min, 32 cycles of (94 26s, 55 40s, 68 3min), 68C 10min, 4C hold
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 7/22 A1 | PCR of part 1 | 7/13 HiFi Part1 |

**Stop Time:** 2:41 am
**Next:** run gel of PCR product

**Date: 7/22/2015
People in lab: Kent Gorday**
**PCR and transformation of HiFi assemblies**
**Start Time:** 2:30pm
**Purpose:** verify and transform assemblies
**Protocol:** LM ed2. PCR: A2 - 10µL MM, 0.5 VR, 0.5 VF, 9 HiFi part 2-1, A3 - 10µL Taq MM, 9µL 50pg/µL trans. eff. kit, 0.5 VR, 0.5 VF2
**Exceptions:** PCR program: 94C 5min, 32 cycles of (94 26s, 58 40s, 68 4min), 68 8min, 4C hold. CTs were performed with 25µL of commercial comp cells
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 7/22 A2 | Part 2-1 pcr'd | HiFi part 2-1, 7/13 |
| 7/22 A3 | positive control for pcr | Trans. eff. kit 50pg/µL |
| 7/22 CT1 | part1 | 7/13 HiFi part1 |
| 7/22 CT2 | part2-1 | 7/13 HiFi part2-1 |
| 7/22 CT3 | part2-2 | 7/13 HiFi part2-2 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/f/f3/Mstigem_jul22_1.jpeg)
![gel electrophoresis](https://static.igem.org/mediawiki/2015/8/84/Mstigem_jul22_2.jpeg)
**Notes:** 7/22 CT4, 7/6 L1, hmp/pro/rbs in amp
**Stop Time:** 9:05pm
**Next:** Run gel of 7/22 A3

**Date: 7/23/2015
People in lab: Kent Gorday**
**Gel of part 2-2 amplified**
**Start Time:** 6:20pm
**Purpose:** verify and amplify HiFis
**Protocol:** LM ed2
**Products:**

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/d/db/Mstigem_jul23_1.jpeg)
![gel electrophoresis](https://static.igem.org/mediawiki/2015/6/6f/Mstigem_jul23_2.jpeg)
**Stop Time:** 8:00pm
**Next:** miniprep, digest and ligate BCs into pSB1C3

**Date: 7/24/2015
People in lab: Kent Gorday**
**Miniprep, PCR, digest of Part 2-1**
**Start Time:** 11:25am
**Purpose:**
**Protocol:** LM ed2, PCR: A1 - 10µL Taq MM, 1µL HiFi part 2-1, 0.25µL VR, 0.25µL 0.1x iGEM VF2, 8.5 water. Digest: D1 - 1µL E, 1µL P, 2.5µL tango, 20.5 µL MP2. Ligation: L1- 1µL buffer, 1µL ligase, 4.6µL ???, 3.4 µL ???
**Exceptions:** PCR program: 94C 5min, 32 cycles (94C 30s, 58C 30s, 68 5.5min), 68C 8min, 4C hold
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 2 | purple ladder 5µL |
| 4 | 7/24 D1 10µL |
| 6 | 7/24 A1 10µL |

**Results:** MP1 - [DNA]: 50, 260/280: 1.83, 260/230: 3; MP2 - [DNA]: 70, 260/280: 1.89, 260/230: 1.8
**Stop Time:** 6:20pm
**Next:** re-do hifi assemblies, transform 7/24 L1

**Date: 7/25/2015
People in lab: Kent Gorday**
**transformation of final pro/rbs/hmp**
**Start Time:** 5:35pm
**Purpose:** clone final hmp/pro/rbs
**Protocol:** LM ed2
**Exceptions:** Commercial comp cells used
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/25 CT1 | hmp/pro/rbs | 7/24 L1 | 2 |

**Stop Time:** 8:30pm
**Next:** Inoculate liquid broth and miniprep

**Date: 7/26/2015
People in lab: Kent Gorday**
**Inoculation of liquid cultures of hmp/pro/rbs**
**Start Time:** 3:10pm
**Purpose:** clone final hmp/pro/rbs
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/26 BC1 | hmp/pro/rbs in chlor | 7/25 CT1 | 2 |

**Stop Time:** 3:25pm
**Next:** Miniprep, digst and gel to confirm

**Date: 7/27/2015
People in lab: Kent Gorday**
**Miniprep, digest of final part pro/rbs/hmp**
**Start Time:** 7:30pm
**Purpose:** obtain final hmp part
**Protocol:** Kitless miniprep protocol, Digest: D1 - 20.5µL 7/27 MP1, 2.5 tnago buffer, 1µL E, 1µL P
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 7/27 MP1, 2 | hmp/pro/rbs in chlor | 7/26 BC1, 2 | 2 |
| 7/27 D1 | hmp/pro/rbs in chlor | 7/27 MP1 | 1 |

**Results:** MP1 - [DNA]: 100, 260/280: 1.71, 260/230: 1.3; MP2 - 100, 1.6, 1
**Stop Time:** 9:30pm
**Next:** run gel of 7/27 D1

**Date: 7/28/2015
People in lab: Kent Gorday**
**Gel of final hmp/pro/rbs**
**Start Time:** 11:30 am
**Purpose:** verify identity of final hmp/pro/rbs
**Protocol:** LM ed2
**Products:**

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/a/a8/Mstigem_jul28.png)
**Stop Time:** 1:30pm
**Next:** Retry electrocomp cells (hmp part is correct)

**Date: 8/18/2015
People in lab: Kira Buckowing**
**PCR of Ocimene synthase**
**Start Time:** 5:25pm
**Purpose:** Amplify gblock from IDT to digest/ligation
**Protocol:** LM ed2, digests: D1 - 12.5µL Taq MM, 5.5 water, 5µL ocimene synthase, 1µL VF2, 1µL VR
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| Ocimene PCR 8/18 | Pcr'd ocimene synthase | Ocimene Synthase 6/30 |

**Stop Time:**
**Next:** Digestion and ligation for transformation

**Date: 8/18/2015
People in lab: Kira Buckowing**
**Digest and ligation of PCR'd ocimene sythase**
**Start Time:** 8pm
**Purpose:** To transfer ocimene synthase gene to chlor BB
**Protocol:** LM ed2. Digest: D1 - 7µL DNA, 1µL P, 1µL E, 2.5 buffer, 13.5 water. Ligations: L1 - 2µL pSB1C3, 6µL D1, 1µL buffer, 1µL ligase; L2 - 4µL pSB1C3, 4µL D1, 1µL buffer, 1µL ligase
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| D1 8/18 | digested amplified gblock | Ocimene Synthase PCR |
| L1 8/18 | ligated ocimene + chlorBB | D1 + pSB1C3 |
| L2 8/18 | ligated ocimene + chlorBB | D1 + pSB1C3 |

**Stop Time:** 10pm
**Next:** Transform ligations

**Date: 8/19/2015
People in lab: Kira Buckowing**
**HiFi Control and transformations**
**Start Time:** 2:45pm
**Purpose:** To ensure quality of HiFi kit, to trnasform ligations into e. coli
**Protocol:** HiFi as per HiFi Manual, Transformations as per LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| HiFi Control 8/19 | HiFi Control | HiFi Kit NEB | 1 |
| CT1 8/19 | HiFi Control | HiFi Control 8/19 | 1 |
| CT2, 3 8/19 | Ocimene synthase | L1, L2, 8/18 | 4 |

**Notes:** Placed plates in G6A fridge 6:30pm 8/20 -LP
**Stop Time:** 5:20pm
**Next:** Check for success, make glycerol stocks or retry

**Date: 8/24/2015
People in lab: Kira Buckowing**
**BC Inoculations**
**Start Time:** 5pm
**Purpose:** To grow cultures from plate colonies
**Protocol:** LM Ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| BC1, 2 8/24 KKB | Possible ocimene synthase | CT2 8/19 | 2 |
| BC3, 4 8/24 KKB | Possible ocimene synthase | CT3 8/19 | 2 |

**Stop Time:** 5:20pm
**Next:** Minipreps and nanodrops

**Date: 8/25/2015
People in lab: Kira Buckowing**
**Minipreps**
**Start Time:** 6:30pm
**Purpose:** Purify plasmids
**Protocol:** Kitless miniprep procedure
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| MP1 8/25 | Poss ocimene synthase. [DNA] 1167, 260/280: 1.73 260/230: 2.47 | BC4 8/24 |
| MP2 8/25 | Poss ocimene synthase. 1411, 1.92, 2.47 | BC2 8/24 |
| MP3 8/25 | Poss ocimene synthase. 929, 1.72, 2.42 | BC3 8/24 |

**Stop Time:** 8:15pm
**Next:** Digest and check for correct

**Date: 8/26/2015
People in lab: Kira Buckowing**
**Digests and gel of ocimene synthase**
**Start Time:** 3pm
**Purpose:** To verify ocimene synthase DNA
**Protocol:** LM ed2. D1 - 2.5 MP1, 2.5 buffer, 1 E, 1 P, 18 water; D2 - 2 MP2, 2.5 buffer, 1 µL E, 1 P, 18 water; D3 - 3µL MP3, 2.5 buffer, 1 E, 1 P, 18 µL water
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 1 | ladder |
| 2 | D1 |
| 3 | D2 |
| 4 | D3 |
| 5 | ladder |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/c/c8/Mstigem_aug26.jpeg)
**Stop Time:** 6:00pm
**Next:** try again

**Date: 9/1/2015
People in lab: Kira Buckowing**
**Modified HiFi attempt**
**Start Time:** 6:20pm
**Purpose:** Attempt to overcome high temp secondary structure in overhang regions of gblocks
**Protocol:** HiFi assembly protocol. Mixed 1-1 2.6µL, 1-2 3.1µL, 1-3 3.1µL, 1-4 3.6 µL, MM 13.7µL
**Exceptions:** Instead of 1hr at 50C, 25 min @ 50C, 10 min @ 60, 15 min @ 50C
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| Mod HiFi1 9/1 KKB | possibly assembled Part1 | part 1 parts, BB |

**Stop Time:** 7:30pm
**Next:** PCR to test for assembly

**Date: 9/2/2015
People in lab: Levi Palmer**
**Streaking pGEX4T-1 cells**
**Start Time:** 4:00pm
**Purpose:** To grow cells containing the pGEX4T-1 plasmid for GST tagging
**Protocol:** 1. Wet sterile swab with LB, 2. Rub swab on bacterial culture, 3. Streak swab on new amp plate. Inoculate BC from swab.
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 8/2/15 pGEX4T-1 | 18|19 | cells containing pGEX4T-1 | pGEX 4T-1 18|19 box1 1/14 |
| 8/2/15 pGEX4T-1 B 18|19 | cells containing pGEX4T-1 | pGEX 4T-1 18|19 box1 1/14 |

**Stop Time:** 5pm
**Next:** Miniprep

**Date: 9/2/2015
People in lab: Levi Palmer**
**PCR and gel of hifi product and ocimene synthase**
**Start Time:** 5pm
**Purpose:** Verify assembly of hifi, amplify ocimene synthase for standard assembly
**Protocol:** LM ed2. PCRs: A1 - 10.5 µL water, 12.5 Taq MM, 0.5 µL VR, 0.5 µL VF2, 1µL 5x10^9 diluted 9/1 hifi; A2 - 10.5 µL water, 12.5µL Taq MM, 0.5 µL VF2, 0.5 µL VR, 1µL 2x10^6 diluted MP2; A3 - 10.5µL water, 12.5µL Taq MM, 0.5 µL VR, 0.5 µL VF2, 1µL 0.5 pg/µL RFP DNA
**Exceptions:** PCR program: 94C 30s, 32 cycles of (95 20s, 57 20s, 68 4.25min), 68 5min, 4C hold
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 3 | purple ladder |
| 4 | 9/2 A1 |
| 5 | 9/2 A2 |
| 6 | 9/2 A3 |

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 9/2 A1 | Part 1 top pathway amplified from HiFi assembly | 9/1 HiFi 1 |
| 9/2 A2 | ocimene synthase amplified from miniprep | 8/25 MP2 |
| 9/2 A3 | RFP pos control | 0.5 pg/µL Trans Eff kit 2015 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/2/26/Mstigem_sep2.jpeg)
**Stop Time:** 10:05pm
**Next:** Check ocimene pcr from 8/18

**Date: 9/3/2015
People in lab: Kent Gorday**
**Gel of ocimene pcr product**
**Start Time:** 10:00am
**Purpose:** verify ocimene pcr
**Protocol:** LMed2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 3 | purple ladder |
| 4 | 8/18 ocimene pcr |
| 5 | 8/18 D1 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/5/5d/Mstigem_sep3.jpeg)
**Stop Time:** 11:35am
**Next:** Determine why pcr failed

**Date: 9/4/2015
People in lab: Levi Palmer**
**Verification of 8/28 ocimene digests**
**Start Time:** 10:30am
**Purpose:** To verify presence of DNA band from 8/26 digests of ocimene synthase
**Protocol:** LM ed2. Digests, D1 - 1000ng DNA, D2 - 2000ng, D3-3000ng, D4 - 2000ng, D5-1000ng, D6-2000ng, D7-3000ng, D8-2000ng. Rest of volume: 2.5µL 10x buffer, 1µL E, 1µL P, filled to 25µL with water
**Exceptions:** D4, D8 had no E or P
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| LP 9/4 D1, D2, D3 | MP2 digested with E&P at various DNA concentrations | MP2 8/25 KKB | 3 |
| LP 9/4 D5, D6, D7 | MP3 digested with E&P at various DNA concentrations | MP3 8/25 KKB | 3 |
| LP 9/4 D4, D8 | Negative controls (No E or P) | MP2, MP3 | 1 |

**Notes:**Well: 1 - D1, 2 - D2, 3- D3, 4-D4, 5-Ladder, 6-empty, 7-D5, 8-D6, 9-D7, 10-D8
**Stop Time:** 3pm
**Next:** Figure out why PCR failed

**Date: 9/9/2015
People in lab: Kent Gorday**
**PCR amplificaton of ocimene synthase, BCs of pGEX4T-1**
**Start Time:** 2pm
**Purpose:** amplify ocimene synthase
**Protocol:** LM ed.2, PCRs: A1 - 10.5 water, 12.5 TaqMM, 0.5 VF2, 0.5 OcimeneR, 1µL 10^5 diluted ocimene gblock. Digest: D1 - 20 water, 2.5µL tango, 1 E, 1P 0.5 A1
**Exceptions:** PCR program: 95C 30s, 32 cycles (95C 20s, 57C 20s, 68C 2min), 68 5min, 4 hold
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 4 | 5µL ladder |
| 5 | 10µL 9/9 A1 |

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 9/9 A1 | amplified ocimene synthase | Part 3glock | 1 |
| 9/9 D1 | nothing | 9/9 A1 | 1 |
| 9/9 BC1 | pGEX4t-1 in amp | pGEX4t-1 | 3 |
| 9/9 A2 | amplified ocimene synthase | part3 gblock | 1 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/5/51/Mstigem_sep9_1.jpeg)
**Notes:** Placed in red freezer box 8:00pm LP 9/9
**Stop Time:** 6pm
**Next:** run gel of A2

**Date: 9/9/2015
People in lab: Kent Gorday**
**Gel of ocimene synthase PCR**
**Start Time:** 8pm
**Purpose:** verify PCR product
**Protocol:** LM Ed2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 5 | ladder |
| 6 | 10µL 9/9 A2 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/b/b0/Mstigem_sep9_2.jpeg)
**Stop Time:** 9:30pm
**Next:**

**Date: 9/10/2015
People in lab: Levi Palmer**
**PCR of ocimene synthase gblock**
**Start Time:** 5:30pm
**Purpose:** to amplify gblock for digestion
**Protocol:** LM ed2, A1 - 10 water, 12.5 MM, 0.5 VF2, 0.5 ocimeneR, 1.5 gblock; A2 - 9 water, 12.5 MM, 0.5 VF2, 0.5 ocimeneR, 2.5 µL gblock
**Exceptions:** ran with igem/hmp pcr program
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| A1 9/10 LP | PCR amplified ocimene gblock | ocimene gblock |
| A2 9/10 LP | PCR amplified ocimene gblock | ocimene gblock |

**Stop Time:** 6pm
**Next:** Gel to confirm

**Date: 9/10/2015
People in lab: Kira Buckowing**
**Gel of ocimene pcr**
**Start Time:** 8:15pm
**Purpose:** confirm ocimene was amplified
**Protocol:** LM ed2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 3 | A1 LP 9/10 |
| 4 | ladder |
| 5 | A2 LP 9/10 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/e/e9/Mstigem_sep10.jpeg)
**Stop Time:** 9:10pm
**Next:** looks fine, new stuff again

**Date: 9/11/2015
People in lab: Levi Palmer**
**Final digest of ocimene block and bb**
**Start Time:** 7:40pm
**Purpose:** to prepare ocimene for ligation
**Protocol:** lm ed2
**Exceptions:** 13µL final volume(buffer adjusted accordingly)
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 9/11 D1 LP | ocimene digested E&P | ocimene gblock |
| 9/11 D2 LP | chlor bb digested E&P | PSB1C3 2015 linear |

**Stop Time:**
**Next:** Ligation

**Date: 9/11/2015
People in lab: Levi Palmer**
**Final ligation of ocimene**
**Start Time:** continued from prev
**Purpose:** to ligate ocimene into chlorbb
**Protocol:** LM ed2.
**Exceptions:** 1:1, 1:3, 1:5 molar ratios BB:Insert used
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| 9/11 LP L1 | 1:1 chlorbb:ocimene | 9/11 LP D1, D2 |
| 9/11 LP L2 | 1:3 chlorbb:ocimene | 9/11 LP D1, D2 |
| 9/11 LP L3 | 1:5 chlorbb:ocimene | 9/11 LP D1, D2 |

**Stop Time:** 10:20pm
**Next:** transform products and/or pcr

**Date: 9/12/2015
People in lab: Levi Palmer**
**PCR of ligations and gel**
**Start Time:** 11am
**Purpose:** to check for complete oci+bb plasmids in ligations
**Protocol:** LM ed2
**Exceptions:** 0.3 µL of ligations used
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 2 | A1 |
| 3 | A2 |
| 4 | ladder |
| 5 | A3 |

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| LP 9/12 A1 | pcr of L1 | 9/11 L1 |
| LP 9/12 A2 | pcr of L2 | 9/11 L2 |
| LP 9/12 A3 | pcr of L3 | 9/11 L3 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/0/0c/Mstigem_sep12.png)
**Stop Time:**
**Next:** gel extraction

**Date: 9/12/2015
People in lab: Levi Palmer**
**GE of ocimene synthase, PCR, gel**
**Start Time:** cont from prev
**Purpose:** To purify pcrs from gel and amplify and confirm
**Protocol:** IBI GE procedure, LM ed2
**Exceptions:** A4 - 2µL GE used, A5 - 4, A6 - 8, A7 - 4. A7 neg control, no primer
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 2 | A4 |
| 3 | A5 |
| 4 | ladder |
| 5 | A6 |
| 6 | A7 |

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| LP GE1 9/12 | combined, gel purified ocimene synthase dna | A1, A2, A3 9/12 LP | 1 |
| LP A4, A5, A6 | amplified ocimene synthase | GE1 LP 9/12 | 3 |
| LP 9/12 A7 | neg control, no primer | GE1 LP 9/12 | 1 |

**Results:** extensive smearing
**Notes:** GE has high salt conc.
**Stop Time:**
**Next:** Retry PCR of L1,2,3

**Date: 9/12/2015
People in lab: Levi Palmer**
**PCR of GE1 and L1 L2 L3, pcr purification of A9**
**Start Time:** cont from prev
**Purpose:** To amplify ocimene synthase, IBI PCR purification protocol
**Protocol:** LM ed2
**Exceptions:** A8 - 1µL L1, A9 - 5/5µL L2/L3, A10 - 2µL L2, A11 - 1 L2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 3 | A11 |
| 4 | ladder |
| 5 | A8 |
| 6 | A9 |
| 7 | A10 |

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| PCR Pure LP 9/12 | ocimene synthase pcr amplified product | LP 9/12 A9 |

**Results:** smearing
**Stop Time:**
**Next:** PCR

**Date: 9/12/2015
People in lab: Levi Palmer**
**PCR of L1, L2, L3 and pcr pure 9/12 and dilution of primers, gel**
**Start Time:** cont. from prev
**Purpose:** To amplify ocimene synthase
**Protocol:** VF2 - 0.16 mg+2.56 ml water, VR - 0.17 mg + 2.78ml water. LM ed2
**Exceptions:** A12 - 0.3 µL L2, A13 - 0.6 µL L2, A14 - 0.3µL L2, A15-0.3µL PCR Pure. A13 and A14 are double volume(MM adjusted accordingly)
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 1 | A12 |
| 2 | A13 |
| 3 | A14 |
| 4 | ladder |
| 5 | A15 |

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| LP 9/12 A12,13,14 | amplified ocimene synthase | L2 LP 9/12 | 3 |
| LP 9/12 A15 | amplified ocimene synthase | PCR pure LP 9/12 | 1 |
| VF2 LP 9/15 | 10uM VF2 primer | IDT VF2 | 2 |
| VR LP 9/15 | 10uM VR primer | IDT VR | 2 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/f/fa/Mstigem_sep13_1.png)
**Notes:** primers in both red and clear freezer boxes
**Stop Time:** 1:30 am
**Next:** Final PCR attempt, CT of L1, L2, L3

**Date: 9/12/2015
People in lab: Levi Palmer**
**Final PCR of ocimene synthase**
**Start Time:** cont. from prev
**Purpose:** Final PCR of ocimene synthase to eliminate smearing
**Protocol:** LM ED2
**Exceptions:** ALC - 0.2 µL L1 used
**Products:**

|  |  |  |
| --- | --- | --- |
| Sample Label | Description | Source Label |
| ALC LP 9/12 | amplified ocimene synthase | L1 9/12 LP |

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| 9/13 CT1,4,5 | ocimene synthase cells? | L1 LP 9/11 | 6 |
| CT2 9/13 | ocimene synthase cells? | L2 LP 9/11 | 2 |
| CT3 9/13 | ocimene synthase cells? | L3 LP 9/11 | 2 |
| BC1,2,3,4,5 | 3µL CT broth in LB+chlor | CT1,2,3,4,5 | 5 |

**Stop Time:** 2:30am
**Next:** gel

**Date: 9/13/2015
People in lab: Kira Buckowing**
**CT of L1-3 9/12 LP**
**Start Time:** 4:30pm
**Purpose:** To check for complete plasmids
**Protocol:** NEB chemical comp protocol
**Exceptions:** Used chemical comp cells
**Stop Time:** 7:20pm
**Next:** Hope for miracle

**Date: 9/14/2015
People in lab: Kira Buckowing**
**BCs of CTs**
**Start Time:** 5pm
**Purpose:** Grow cultures for MPs
**Protocol:** LM ed2
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| BC 1, 2, 3, 4, 5 | BC of ocimene synthase? | CT2 | 5 |
| BC 6, 7, 8, 9, 10, 11 | ocimene synthase? | CT3 | 5 |
| BC 12-21 | ocimene synthase? | CT5 | 10 |

**Stop Time:** 5:50pm
**Next:** miniprep anything that grows

**Date: 9/15/2015
People in lab: Kira Buckowing**
**Minipreps**
**Start Time:** 1:30pm
**Purpose:** Purify a plasmid of ocimene synthase
**Protocol:** Kitless miniprep procedure
**Products:**

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| MP1-15 | is it ocimene synthase? | BC1-15 | 15 |

**Stop Time:** 7:30pm
**Next:** Digest with e&p, run gel

**Date: 9/15/2015
People in lab: Kira Buckowing**
**Digest and gel**
**Start Time:** 7:50pm
**Purpose:** to check for ocimene synthase (1750bp)
**Protocol:** LM ed2
**Products:**

|  |  |
| --- | --- |
| Well | Sample |
| 1 | D1/D9 |
| 2 | D2/D10 |
| 3 | D3/D11 |
| 4 | D4/D12 |
| 5 | L/L |
| 6 | D5/D13 |
| 7 | D6/D14 |
| 8 | D7/D15 |

|  |  |  |  |
| --- | --- | --- | --- |
| Sample Label | Description | Source Label | Quantity |
| KKB D1-15 9/15 | Ocimene synthase cut E&P? | MP1-15 9/15 KKB | 15 |

**Results:**
![gel electrophoresis](https://static.igem.org/mediawiki/2015/6/6f/Mstigem_sep15_1.jpeg)
![gel electrophoresis](https://static.igem.org/mediawiki/2015/3/3b/Mstigem_sep15_2.jpeg)
**Stop Time:** 9:40PM
**Next:** This is the end.

Missouri cave photo by Lynn Dieter