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From 2011.igem.org

July 10th - July 16th

Sunday, July 10

Lab work done by: James Mathew, Charlie Chung

• Worked on website

- Preliminary design of banner, safety content, and temporary lab notebook spreadsheet

• Ligation product was transformed in electrocompetent cells
• Culture plated on agar plate treated with ampicillin
• Plasmid containing the Vio operon was transformed and plated on agar treated with kanamycin
• Overnight PCR reaction for RFP

Monday, July 11

Tuesday, July 12

• Finished final design of team logo

Wednesday, July 13

• "Website Team" meeting to continue design and construction of website

- Designed and constructed final banner design

Thursday, July 14

Lab work done by: James Mathew, Charlie Chung

• Sequencing revealed no GFP in the ligation product
• Will retry the 3-piece ligation of GFP + Annealed Primer Pair A (half of biotin tag) + Annealed Primer Pair B (other half of biotin tag & iGEM suffix)
• PCR to amplify vector backbone containing RFP
• Met Prof. Lucks -- prospective iGEM advisor and coolest man alive

Friday, July 15

• Banner was updated to include Flash animation of moving gears.

Team Meeting

1. Ligation didn't work -- retry over the weekend

   - transform and plate today

   - inoculate and colony PCR on Saturday

   - Miniprep and prepare for sequencing on Sunday

2. Prepare more culture of backbone plasmid

   - inoculate today

   - Miniprep on Saturday

3. Order primers today for other methods of ligation: (1) longer primers (2) two-step PCR with shorter primers
4. Order primers for our pathway enzymes: VioA, VioB, VioB

   - Primers should come Wednesday

   - If ligation this weekend fails, we can try these other PCR methods

5. Animations and website construction are continuing

Lab work done by: James Mathew, Charlie Chung, Youjin Cho

• Running low on pZE vector backbone, so fired up two cultures
• Miniprep to purify out the plasmid. Followed standard Qiagen Miniprep protocol for microcentrifuge
• Verify if PCR amplification of RFP worked using gel electrophoresis. If yes, then proceed to gel extraction and purification

- NOTE: Thermocycler did not keep our rxn tube at 4°C

• Electroporation method of transforming our 3-piece ligation product and control into DH5α (competent E. coli = made porous for uptake of outside DNA)
• Plated transformed bacteria onto agar plates treated with ampicillin

Saturday, July 16

Lab work done by: Youjin Cho, Nicholas Kramer

• Growth of numerous colonies suggests the ligation reaction worked

- Will pick 2 colonies off the plate in the evening and inoculate overnight

• PCR to amplify vector backbone containing RFP

- Melting temperature of 57°C leads to non-specific binding

- Ran 2 PCR reactions with melting temperatures 59°C and 61°C