User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/21

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χρόνος πέρασμα September 21th 2011

HydA CAI Optimization Control

ABSTRACT
  • PCR of the HydA optimized CDS using Pfx platinum polymerase from Life Technologies and the PCRx Enhancer 10x Buffer.

  • Today I tried another strategy to perform the optimized HydA CDS that I need to proceed with the assembly of the construction that will serve to test if the CAI optimization works properly. I used a Pfx platinum polymerase from Life Technologies as well as a special buffer called PCRx Enhancer 10x Buffer, that is specially used for primers that are long and/or rich in GC. The primers I'm using are both; they are 34 nt long and 61 nt long, with a GC content of 50% and 48%, respectively. The manufacturer's advise is that this buffer should be tried at different concentrations (e.g. 0.5x, 1x, 2x) [1]. So I prepared three different sets of reactions that varied with this buffer, and the same set was subjected to a temperature gradient to see what annealing temperature gives the best amplification (and also to test if any secondary structure or primer dimers inferfere with the reaction, greater the temperature, less secondary structures). I also prepared reactions to amplify the wild-type HydA CDS, this served as a positive control, as I have already been able to amplify this region.
  • The reactions were prepared as follows:


Reactants PCRx Enhancer Buffer 0.5x PCRx Enhancer Buffer 1x PCRx Enhancer Buffer 2x
H20 32.9 μl 30.4 μl 25.4 μl
Pfx Buffer 10x 10 μl 10 μl 10 μl
8mM dNTPs mixture 1.7 μl 1.7 μl 1.7 μl
25mM MgSO4 2 μl 2 μl 2 μl
Forward primer 2 μl 2 μl 2 μl
Reverse primer 2 μl 2 μl 2 μl
DNA template 1.5 μl 1.5 μl 1.5 μl
PCRx Enhancer 10x 2.5 μl 5 μl 10 μl
Pfx platinum Pol 0.4 μl 0.4 μl 0.4 μl
Total 50 μl 50 μl 50 μl


  • Incubation 5 min 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, I ran a 1% agarose electrophoretic gel to see if there was in fact some amplification. 130 V 35 minutes, 5μl from each PCR tube was used in each well.


Well PCR product
1 250 bp Ladder
2 HydA NoOpt PCRx Enhancer Buffer 0.5x
3 HydA NoOpt PCRx Enhancer Buffer 1x
4 HydA NoOpt PCRx Enhancer Buffer 2x
5 HydA Opt Enhancer Buffer 0.5x 55°C
6 HydA Opt Enhancer Buffer 0.5x 61°C
7 HydA Opt Enhancer Buffer 0.5x 68°C
8 HydA Opt Enhancer Buffer 1x 55°C
9 HydA Opt Enhancer Buffer 1x 61°C
10 HydA Opt Enhancer Buffer 1x 68°C
11 HydA Opt Enhancer Buffer 2x 55°C
12 HydA Opt Enhancer Buffer 2x 61°C
13 HydA Opt Enhancer Buffer 2x 68°C



  • As it can be seen, the positive controls did amplified. All these three (wells #2,#3 and #4) were subjected at the same annealing temperature (the one that proved the best in the last PCR, 55°C); it should be noted that a greater concentration of the PCRx Enhancer Buffer inhibited the amplification. None of the other wells show any amplification :(


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