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:
Reactants
Volume
H20
34 μl
PCR Buffer 10x
5 μl
0.4mM dNTPs mixture
4 μl
50mM MgCl2
1 μl
Primer forward 5μmol
2 μl
Primer reverse 5μmol
2 μl
DNA template
1.5 μl
Taq platinum Pol
0.5 μl
Total
50μl
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.
Well
PCR reaction
1
500 bp Ladder
2
HydAOpt 55°C
3
HydAOpt 63°C
4
HydAOpt 72°C
5
HydA NonOpt 55°C
6
HydA NonOpt 63°C
7
HydA NonOpt 72°C
8-12
Other Stuff
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...
UNAM Genomics Mexico 2011
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