Team:Imperial College London/Wetlab/Protocols/PCR

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(New page: =PCR Protocol= ==Reaction Mixture == Each PCR tube should contain: (25ul total) * 2.5ul of 10x Pfu Ultra Buffer * 0.5ul dNTPs * 1ul Primer Fwd (100ng) * 1ul Primer Rev (100ng) * 0.5ul P...)
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Revision as of 12:32, 30 September 2009

PCR Protocol

Reaction Mixture

Each PCR tube should contain: (25ul total)

  • 2.5ul of 10x Pfu Ultra Buffer
  • 0.5ul dNTPs
  • 1ul Primer Fwd (100ng)
  • 1ul Primer Rev (100ng)
  • 0.5ul Pfu Ultra
  • 1ul DNA Template

This can be made as a master mix, to reduce variation between reaction mixtures. Master Mix contents (for 4 PCR tubes):

  • 12.5ul Pfu Buffer
  • 2.5ul dNTPs
  • 5ul Primer Fwd (500ng)
  • 5ul Primer Rev (500ng)
  • 2.5ul Pfu Ultra

Pipette 24ul of the master mix into different PCR tubes. To each experimental tube add 1ul DNA template, and to the negative control, add 1ul ddH2O.


Cycles

X = Annealing Temperature (X1 temp without overhang, X2 temp with overhang), Y = Length of contruct to work out elongation time (for Pfu Ultra II, the rate is 15sec per kB, minimum 15 seconds)

1 cycle: 2 minutes at 95'C
10 cycle: 30 seconds at 95'C, 45 seconds at X'C, Y seconds at 72'C 20 cycle: 30 seconds at 95'C, 45 seconds at X2'C, Y seconds at 72'C 1 cycle: 5 minutes at 72'C

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