User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/19

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PCR attempt

Aim: To amplify the CHD gene using P2/P8 primers.

Method: I tried to follow the protocal listed in the paper that I initially read that uses P2/P8 primers. We used the following concentrations in the tubes

PCR reaction tube:

  • 2.5ul Forward primer
  • 2.5ul Reverse Primer
  • 2.0ul DNA
  • 1.5ul MgCl2
  • 1.5ul PCR Buffer
  • 2.5ul dNTPs
  • 1U Taq polymerase

to 25ul H2O

The dNTPs were not added until after the PCR had started. They were added and the reaction was restarted. PCR settings were:

  • 94C for 1m 30s
  • 95 30s
  • 52 30s
  • 72 30s
  • 72 5min

This may not be accurate as the reaction was restarted at 66C. This shouldn't be too much of a problem though. The reaction may not work because the fidelity of the Taq may have been reduced due to the reaction going twice.

P2/P8 primers were diluted to make a 100mM/L stock and then a 10mM/L stock was made to have a final dilution of 1:10. When 1ul of the primer is added to 10ul of PCR mix, this is the desired concentration. Also, I used 1U of Taq instead of the 0.5U suggested in the original protocol.

One of the reaction tubes may have had too much master mix added. I marked this tube with an X.

Results: To be determined via agar gel electrophoresis.