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Team:UTK-Knoxville/28 June 2013
From 2013.igem.org
June 28th, 2013
We now have stock of Azo-Tray 14, Box 1, Well 53. Patrick - Set up a 25 uL Q5 master mix PCR reaction to extract HemAT from the R. Leg gDNA. Used HemAT_F and HemAT_R with a Tm of 52 and 56, an elongation time of 1:15 minutes, and 32 cycles. This is the Tm established by our analysis with APE when creating the primers. The Tm from the sides of the primer tubes would suggest a Tm of 60 C but this is taking into account the full length of the primer (including the overhang). HemAT is 558 bp and the master mix has an extension of 30 seconds / kb. 30-35 cycles is recommended for gDNA amplification. I used 2 uL of template DNA (~50 ng). Tomorrow Eric will run a gel and isolate HemAT. I planned to make stock of R. Leg today, but it did not grow while I was gone, so I am growing more in the 37C shaker and will make stock Monday. Brandon ran a gel on Patrick’s PCR of HemAT and RcoM. The PCR did not work, so he has set up a PCR at a range of temps(56, 60, 64) see if the low temperature is causing the problem.
