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Team:Cornell/Week 16
From 2011.igem.org
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September 18th - September 24th
Sunday, September 18
Monday, September 19
Lab work done by: Jim Mathew, Youjin Cho, Charlie Chung
- Background
- The PCR site-directed mutagenesis done earlier today should ideally enhance protein expression of VioA, VioB, VioE, and RFP by deleting excessive nucleotides between the ribosome binding site and the ATG start codon in the original DNA sequences
- Work Accomplished
- PCR procedure for deletion technique
- Using 1µl of PCR product of VioA, VioB, VioE and RFP, transformed them into DH5α electrocompetent cells via electroporation
- Put the transformed samples in 37°C shaker for an hour
- Plated the transformed products of VioA, VioB, VioE, and RFP on agar plates treated with ampicillin or carbenicillin (two are on amp; other two are on carb)
- Incubating at 37°C in Olin 303
Tuesday, September 20
Afternoon lab work done by: Charlie Chung
- VioA, VioB, and VioE plates = lots of bacterial colonies, high-density growth
- - Picked three colonies from each plate and started up (3mL LB + 3µL carbenicillin) cultures
- VioA-1,2,3
- VioB-1,2,3
- VioE-1,2,3
- - Incubating at 37°C in Olin 304
- RFP plate = ~5 colonies
- - Picked two colonies and started up (3mL LB + 3µL carbenicillin) cultures
- RFP-1,2
- - Incubating at 37°C in Olin 304
Wednesday, September 21
Morning lab work done by: Charlie Chung
- Transferred 500µL from each culture tube to 20mL (LB + carbenicillin) subcultures
- - Shaking at 37°C in Olin 304
- Miniprep the remaining cultures of VioA-1,2,3 ; VioB-1,2,3 ; VioE-1,2,3 ; RFP-1,2
- - Because of time constraint, was not able to quantify the DNA concentration
- - Sitting in the freezer of Olin 301
Afternoon lab work done by: Nancy Li, Youjin Cho
- After 5 hours of subculturing, transferred all the samples into corresponding 50mL tubes and spun them down at 4500rpm for 30 minutes
- Poured off the supernatant and stored the pellets at -20°C for future use
Thursday, September 22
Afternoon lab work done by: Charlie Chung
- Quantified the Miniprep-purified products with NanoDrop
- - VioA-1 = 68ng/µL
- - VioA-2 = 60.2ng/µL
- - VioA-3 = 88.1ng/µL
- - VioB-1 = 117.6ng/µL
- - VioB-2 = 89ng/µL
- - VioB-3 = 133.6ng/µL
- - VioE-1 = 80.5ng/µL
- - VioE-2 = 73.6ng/µL
- - VioE-3 = 77.4ng/µL
- - RFP-1 = 84.4ng/µL
- - RFP-2 = 56.5ng/µL
- Prepared samples for sequencing submission
- Order Number:10257313
