Team:Lambert GA/Notebook
Notebook
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Week 1 (Aug 3):
Since we had been having difficulties working with p-lambda-r LacI, we discussed the merits of using a different promoter for our tsPurple constructs.
Week 2 (Aug 7):
After the previous week's discussions, we transformed, inoculated, and miniprepped weak pLac (hoping that a weaker promoter would cause less strain on our cells). In addition, we attempted to ligate the tsPurples into 1C3, however the miniprep concentrations had been too low, so we needed to order more of our parts from IDT.
Week 3 (Aug 14):
We came to the decision that pLac would not work when our construct was fully assembled because it is the same promoter that is on the second part of our construct. Due to this, we moved back to p-lambda-r LacI and inoculated liquid cultures from a pre-existing plate. In addition, we transformed and then miniprepped our tsPurple constructs, however the concentrations of both p-lambda-r LacI and the tsPurples were shockingly low so we decided to run a PCR next week.
Week 4 (Aug 21):
After last week's low miniprep concentrations we ran PCRs of the tsPurples, p-lambda-r LacI, and pLac-ClpXP-CI. We also discussed our lab work with Dr. Styczynski and Monica McNerney to determine potential sources of error.
Week 5 (Aug 28):
After last week's PCR we did a cleanup and nanodropped, which yielded good concentrations. We then sent p-lambda-r LacI and R0011ClpXPCI for sequencing and ligated and transformed our tsPurple constructs.
Week 6 (Sep 4): Since the previous transformations were unsuccessful (with concerns about contamination and plates with the wrong antibiotic), we decided to re-transform. Since those were successful, we ran a colony PCR, however the gel showed that the colony PCR was unsuccessful. Meanwhile, we transformed our RFP proof of concepts.
Week 7 (Sep 11): We inoculated liquid cultures of p-lambda-r LacI and retransformed our tsPurple constructs after the unsuccessful colony PCR, however the transformations were unsuccessful as well so we decided to return to earlier minipreps and start over. After starting over and retransforming, it turned out third time is not the charm since it was unsuccessful again. We decided to go another step back and re-digest from the original hydrated tsPurple constructs. Simultaneously, we had an unsuccessful miniprep of p-lambda-r LacI (but a successful one of pLac-ClpXP-CI), so we went back to an old digest and ran all three tsPurple digests and p-lambda-r LacI on a gel. While the tsPurples were the expected base pair length, p-lambda-r LacI did not show up, potentially because of incredibly low concentration.
Week 8 (Sep 18): morp
Week 9 (Sep 25): maybe delete pics?
Week 10 (Oct 2): meep
Week 11 (Oct 9): morp images? delete?
Week 11 Miniprep R0011--ClpXP--CI and Week 11 Miniprep P-Lambda-R-LacI--GFP--ClpXP-CI
Week 13 (Oct 23): meep
Week 14 (Oct 30): meep
