User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/11

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χρόνος πέρασμα September 11th 2011

HydA CAI Optimization Control

  • PCR of the optimized HydA CDS using Taq platinum and a different dilution of the plasmid template.

  • Today, I repeated the PCR that is supposed to amplify the optimized HydA CDS that will serve to prove if the codon optimization that was performed on the sequences we sent synthesize did make a difference for Rhizobium etli CFN42 in its growth. As the past PCR was not succesful, I now tried to amplify from another stock of the plasmid, one that was cutted with restriction enzymes (is linearized), extracted from an E.coli transformation. I used Taq platinum from Invitrogen.
  • The reactions were prepared as follows:

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 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 test the amplification. 130 V 35 minutes. As a positive control, I used the primers and the template to amplify the wild-type HydA.

Dramirez PCR Helendilution taqplatinum.jpg

  • As it can be seen, there was no amplification for neither hydrogenases CDS. Neither this plasmid nor the other I've been using appear to work properly for amplification.