User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/20

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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 [1].
  • 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:

Reactants Volume
H20 34 μl
Pfx 10x Buffer 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
Pfx platinum Pol 0.5 μl
Total 50μl

  • 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.

Well PCR reaction
1 250 bp Ladder
2 HydAOpt 55°C
3 HydAOpt 61°C
4 HydAOpt 68°C
5 HydANonOpt 55°C
6 HydANonOpt 61°C
7 HydANonOpt 68°C

Dramirez PCR Pfxplatinum temperaturegradient HydA.jpg

  • 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.