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Team:Cornell/Week 6
From 2011.igem.org
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June 10th - June 16th
Sunday
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 Amp
- Vio-Operon plasmid was transformed and plated on Kanamyacin
- RFP PCR done overnight
Monday
Tuesday
- Finished final design of team logo.
Wednesday
- Website Meeting to continue design and construction of website. Designed and constructed final banner design.
Thursday
Lab work done by: James Mathew and Charlie Chung
- Sequencing revealed no GFP. 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 adviser and coolest man alive
Friday
- Banner was updated to include flash animation of moving gears.
Team Meeting
- Ligation didn't work, need to retry it over the weekend (transform and plate today, innoculate and colony PCR saturday, miniprep and prepare for sequencing on sunday).
- Prepare more culture of backbone plasmid (innoculate today, miniprep on saturday)
- We want to order primers today! for other methods of ligation, with longer primers, and two step PCR with short primers, in case our current one doesn't work. Also going to order primers for our pathway enzymes. The primers should come Wednesday. If ligation this weekend fails, we can try these other PCR methods.
- Animations and website are continuing
- Running low on pZE vector backbone, so borrowed two culture tubes from Sean. 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 @ 4 deg 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 LB + Ampicillin. Followed protocol as written in the "Multimedia" section.
Saturday
Lab work done by: Youjin Cho
- Looked at the plates for ligation. Ligation worked!! Sean will pick 2 colonies off the plate in the evening and innoculate overnight.
- PCR to amplify vector backbone containing RFP.
- Melting temperature of 57C leads to non-specific binding. We ran 2 PCR reactions with melting temperatures 59C and 61C.
