χρόνος πέρασμα September 13th 2011
HydA CAI Optimization Control
- PCR of the optimized HydA CDS using Taq platinum polymerase in a temperature gradient for the annealing step.
- Once again, I tried to amplify through PCR the optimized HydA CDS. As it has proved difficult to be amplified, I asked one of our professors, Christian Sohlenkamp, if he could help me. He told me that I should try letting the PCR in a temperature gradient for the annealing step to see which temperature was the best to amplify; he also told me to use bioinformatic tools to see if the primers I designed could make secondary structures that hindered the amplification. I checked them, and they did happened to contain certain regions that could prevent them from hybridizing to the DNA template; the temperature gradient should help minimizing this threat. I used a Taq platinum polymerase from Invitrogen.
- I used the following reactants concentrations:
|PCR Buffer 10x
|0.4mM dNTPs mixture
|Primer forward 5μmol
|Primer reverse 5μmol
|Taq platinum Pol
- Incubation 3 minutes at 94°C
- 30 Cycles
- Denature: 30 seconds at 94°C
- Anneal: 55°C-72°C for 30 seconds
- Extend: 1:55 minutes at 72°C
- Post-Incubation: 2 minutes at 72°C
- After the PCR, I ran a 1% agarose electrophoretic gel to see if any amplification was succesful. 130 V 35 minutes.
||500 bp Ladder
||HydA NonOpt 55°C
||HydA NonOpt 63°C
||HydA NonOpt 72°C
- As it can be seen, there was no amplification at all for any of the reactions, even for the ones that I used as positive control (the PCR of the HydA NonOpt, that I have already amplified). No idea why...