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Team:CSU Fort Collins/Notebook/Breakdown/Aug
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Revision as of 17:14, 17 October 2014
Breakdown Daily Notes
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July
Lac Promoter and FadD Assembly
August
PLR, PFL, and PFL2 Assembly
September
Scientists vs. the PCR Machine
AUGUST
Friday, August 1
Discussed with Sun to troubleshoot why the PCR wasn’t working. We discovered that the final base in one of our primers was incorrect. We corrected that and reordered the primer.Tuesday, August 5
Reran PCR with correct primer using previously isolated E. coli genome.Wednesday, August 6
Checked PCR with gel, this gel failed. Reran PCR using a gradient of temperatures. Checked this PCR with a gel, which also failed.Thursday, August 7
Reran Gel with higher concentrations of PCR product. This gel also failed.Friday, August 8
Discussed with Sun why the PCR was not working. Discovered that when finding the melting temperatures we had not used the specifications of the polymerase we were using. Reran the PCR using a gradient around the new melting temperature. We used both colonies and previously isolated E. coli genome in different replicates for our template DNA. Ran a gel to determine success. The conditions which gave us the required 1.7 kb strand were 61 ºC and 1.7 minute extension step for the first five cycles and 72 ºC and 1.7 minute extension step for the last 30 cycles. The template DNA that worked came from the previously isolated E. coli genome.
Gel Results, August 8
