Team:UCLA/Notebook/Protein Cages/28 July 2015
Tried PCR using new forward primer #2, reverse primer #1, and cage with his-tag template. Saw no product whatsoever, just like last time using forward primer #2. Thus, we should probably scrap this primer and not use it for further experiments.
Meeting Notes: Sri suggested that for all PCRs with extensions, we should first use a low melting temp (55-60C) for the first 5 cycles. Then alternate between 72 and 98C for about 20 cycles. The 72C time will function as both the annealing and extension time. He also recommended that we dilute the template DNA, as a concentration that is too high can affect annealing temperature. He said the dNTP and primer concentrations should be kept the same as the normal Q5 protocol. He said we should start with 1 ng per reaction and do 10-fold dilutions to optimize the template concentration.
We will try another gradient PCR for the first 5 rounds, followed by a two-step reaction. According to NEB, the annealing/extension time may be kept the same as the usual 20-30 seconds per kb. The reaction will be started today and ran on a gel tomorrow.
Tyler Lee --Wtleeiv 19:41, 28 July 2015 (CDT)
Nithin's Notes
Ran the PCR suggested by Sri. But no band was seen
Conditions:
- 1 cycle
- 98 degrees-30s
- 5 cycles
- 98 degrees -10s
- 58-66(gradient) degrees - 20s
- 72 degees - 30s
- 20 cycles
- 98 degrees- 10s
- 72 degrees- 45s
- 4 degree hold