User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/13

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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:

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...

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