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

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(Autocreate 2012/11/19 Entry for User:Stuart_McKellar/Notebook/Bird_Sex_Testing)
Current revision (23:57, 18 November 2012) (view source)
(Entry title)
 
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==Entry title==
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==PCR attempt==
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* Insert content here...
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Aim: To amplify the CHD gene using P2/P8 primers.
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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
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PCR reaction tube:
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*2.5ul Forward primer
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*2.5ul Reverse Primer
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*2.0ul DNA
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*1.5ul MgCl2
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*1.5ul PCR Buffer
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*2.5ul dNTPs
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*1U Taq polymerase
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to 25ul H2O
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The dNTPs were not added until after the PCR had started. They were added and the reaction was restarted. PCR settings were:
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*94C for 1m 30s
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*95 30s
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*52 30s
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*72 30s
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*72 5min
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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.
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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.
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One of the reaction tubes may have had too much master mix added. I marked this tube with an X.
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Results: To be determined via agar gel electrophoresis.
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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.


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