χρόνος πέρασμα September 20th 2011
HydA CAI Optimization Control
- PCR of the optimized HydA CDS using Pfx platinum polymerase from Invitrogen and a temperature gradient for the annealing.
- Today I again tried to amplify the optimized HydA CDS but now using a different enzyme, so far, I had been using a Taq platinum from Invitrogen, but now I tried using a Pfx platinum polymerase also from Invitrogen. I followed the manufacturer's protocol .
- The PCR was performed on a temperature gradient that went from 55°C to 68°C in the annealing step. This was to try to determine the best temperature at which the amplification was the highest. I used as a positive control the primers and template DNA that amplify for the wild-type HydA CDS.
- The reactions were prepared as follows:
|Pfx 10x Buffer
|0.4mM dNTPs mixture
|Primer forward 5μmol
|Primer reverse 5μmol
|Pfx platinum Pol
- Incubation 5 minutes at 94°C
- 30 Cycles
- Denature: 94°C for 15 seconds
- Anneal: 55°C-68°C for 30 seconds
- Extend: 68°C for 1:55 minutes
- The reactions were maintained at 4°C once the cycles finished.
- Once finished the PCR, I ran a 1% agarose electrophoretic gel. 130 V 35 minutes, 5μl from each PCR tube were used per each well.
||250 bp Ladder
- As it can be seen, the positive control only was amplified at 55°C. There was no amplification for the optimized HydA CDS. Perhaps the long primers are the culprits.