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Team:UC Davis/Notebook/Week 9
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After many weeks of wetlab, our mutant screening/characterizing constructs are finally done! We have sent them off for sequencing and expect them back next week. We also made more R0010 mutants with our new refined protocol. Hopefully we can start screening next week.
--Monday 8/08/11--
The transformations from Sunday still have a bit of background from the vector but look much more promising than the past few attempts. Just to be certain, we set up another PCR screen and ran the products on a gel. Between all of the PCR screens we have run over the past week, it looks like we finally have all of the parts that we need to begin testing our mutants. We cultured the colonies that looked good and will have them sequenced to make sure that they are the correct parts.
Today we miniprepped the cultured constructs, nanodropped them, and sent them off for sequencing.
Did some more testing of our mutant PCR protocol. Tested various reaction conditions on R0010: perhaps using more template DNA will improve results, or running the PCR multiple times in succession. The reference paper suggested that we can expect between 1 and 20 mutations per kb. Team:UC_Davis/Protocols#Error_Prone
Re-ran PCRs from yesterday to create second-round mutants, using 1/100th dilution of PCR product.
This product was then used to create a third round of mutants.
Mutants were then PCR prepped. Ran a diagnostic gel, got large, smeary, thick bands. All three were digested with EcoRI and SpeI, and the E0240-I13453-B0034-(Repressor) constructs were digested with EcoRI and XbaI.
R0010 PCRs (from all three rounds) were gel purified, as well as J61002.
