Team:Warsaw/Calendar-Main/13 August 2009

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<li>Template DNA in 3 dilutions - 1x10^1 (pure plasmid DNA), 1x10^2 and 1x10^3.</li>
<li>Template DNA in 3 dilutions - 1x10^1 (pure plasmid DNA), 1x10^2 and 1x10^3.</li>
<li>buffer, dNTP mix and polymerase from EURx.</li>
<li>buffer, dNTP mix and polymerase from EURx.</li>
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<p>Thermal cycling conditions for PCR (~7-8 kb):</p>
<p>Thermal cycling conditions for PCR (~7-8 kb):</p>
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Revision as of 12:14, 13 August 2009


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Amplyfing of Bax sequence

Justyna

Methods


  • PCR reaction mix:

  • per 50μl:
    5.0 μl - 10 x buffer (20mM MgSO4)
    3.5 μl - dNTP mix (5mM)
    3.0 μl - primer BaxF (0.5 μM)
    3.0 μl - primer BaxR2 (0.5 μM)
    1.0 μl - plasmid DNA template
    32.0μl - mQ water
    2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl)
    

  • Template DNA in 3 dilutions - 1x10^1 (pure plasmid DNA), 1x10^2 and 1x10^3.
  • buffer, dNTP mix and polymerase from EURx.



  • Thermal cycling conditions for PCR (~7-8 kb):


    94°C, 1 min 0s
    
    94°C, 0 min 15s
    69°C-74°C, 0 min 30s
    68°C, 1 min 0s
    cycles 1-10
    
    94°C, 0 min 15s
    69°C-74°C, 0 min 30s
    68°C, 1 min 20s
    cycles 11-25
    
    68°C, 7 min 0s
    4°C, indefinite
    

    Results:

  • Best reaction for 1x10^1 and 1x10^2 (more specific), in 69.1°C and 69.8°C.

April
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May
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June
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29 30
July
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August
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31
September
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October
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