PCR

Reaction Mixes

Using Vent Polymerase (100 ul reactions):

  • 80.5 ul distilled H2O
  • 10 ul ThermoPol buffer
  • 2 ul 10 mM dNTPs
  • 2 ul 100 mM MgSO4
  • 2 pmoles primer1, usually 2 ul of a 100 pM stock
  • 2 pmoles primer2, usually 2 ul of a 100 pM stock
  • 1 ul miniprep or Qiagen DNA, usually around 200-500 ng template
  • 0.5 ul Vent DNA Polymerase

Using Taq Polymerase (100 ul reactions):
  • 79 ul distilled H2O
  • 1 ul 1 M Tris, pH 8.5
  • 1.5 ul 3 M KCl
  • 5 ul 20% Triton X-100
  • 2 ul 10 mM dNTPs
  • 6 ul 25 mM MgCl2
  • 2 pmoles primer1, usually 2 ul of a 100 pM stock
  • 2 pmoles primer2, usually 2 ul of a 100 pM stock
  • 1 ul miniprep or Qiagen DNA, usually around 200-500 ng template
  • 0.5 ul Taq DNA Polymerase

Note: Volume of distilled H2O can be adjusted if additional volumes of reagents are required.

PCR Cycle (for PE 9600 or equivalent):

95oC for 5 minutes

95oC for 30 seconds
55oC for 1 minute
72oC for 2 minutes
  ==> 35 cycles
72oC for 10 minutes
4oC hold

Note: The incubation times at each temperature can be varied to accomodate different volumes of reactions.

Also, annealing temperature may have to be determined emperically.