Team:Cornell/Week 6

From 2011.igem.org

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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