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Monday 8/2
- Group meeting today!
- Started lots more CPCR:
- 2 colonies each of: L24, L22, HSP-Cm, L20, L31, L49, L48, L26, and M30109 using distribution DNA as template.
- Successful: L24-1/-2, 20-4/-5, L22-1/-2, L48-4
- Started ligations:
- L49: HSP-Amp + L40 + pSB1T3
- L54: HSP-Amp + K215000 + pSB1K3
- L55: K215000 + I13504 + pSB1K3
- Began 2 1mL LB-Kan cultures of 42-4/-5 & incubated @ 30°C
- Began 2 5mL LB-Kan cultures of 43-4/-5 & incubated @ 37°C for sequencing tomorrow.
Tuesday 8/3
- Ligations all failed! (pos control fine)
- Prepped 42-4/-5 & sent out for sequencing
- Prepped 48-4, 20-4/-5, 22-1/-2, 24-1
- Concentrations all over 200ng/μL, so diluted all 2x with diH2O.
- Began colony PCR to verify the contents of the lysis casette and lysis gene (K124017 & K124017)
- After checking the Registry page, we decided that there may be some sort of problem with the DNA or error in its cataloging.
- Also started colony PCR of M30109 cells and ran M30109 digest DNA in the gel to similarly try to verify the presence of the desired insert, after talking with the UNAM-Genomics_Mexico iGEM team.
- Digested L23, L44, L22, L24, more Cm and Kan backbone, and a few other bricks
- Began ligations:
- L36: K215000 + L22 + pSB1K3
- L38: L19 + L22 + pSB1A3
- L37: L23 + L24 + pSB1K3
- L56: K124017 + B0015 + pSB1C3 (will ligate Thurs)
- L57: K124014 + B0015 + pSB1C3 (will ligate Thurs)
Wednesday 8/4
- All ligation plates were successful (even though they were 20 min only)
- Began colony PCR (x3) on each: L37, L49, L54, L55
Thursday 8/5
Friday 8/6
Weekend 8/7-8/8
- Used ligation of HSP + GFP to test temperatures at which transcription occurs.
- Tested fluorescences at 4˚C, 25˚C, 37˚C, 42˚C, and 45˚C.
- Fluorescence measured increased as temperature increased - a sign that HSP works.
- Even at the low temperature of 4˚C, there was a significant amount of fluorescence.
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