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.