Team:CU Boulder/Protocols/PCR


<!DOCTYPE html> Team:CU_Boulder - 2015.igem.org

PCR Methods

Reaction components

Q5 Reaction

Reagent50 ul reactionFinal Concentration
5x Q5 buffer10 ul1x
10 mM dNTPs1 ul200 uM
10 uM F. Primer2.5 ul0.5 uM
10 uM R. Primer2.5 ul0.5 uM
DNA1 pg - 1 ng< 1,000 ng
Q5 Polymerase0.5 ul0.02 U/ul
Waterto 50 ul

Phusion Reaction

Reagent50 ul reactionFinal Concentration
5x Phusion buffer10 ul1x
10 mM dNTPs1 ul200 uM
10 uM F. Primer2.5 ul0.5 uM
10 uM R. Primer2.5 ul0.5 uM
DNA1 pg - 10 ng< 250 ng
Q5 Polymerase0.5 ul1 U/5ul
Waterto 50 ul

Reaction Conditions

Notes:

  • Annealing temperature depends on primers. To determine the optimal annealing temperature, use the NEB Tm calculator
  • Extension time depends on length of amplicon. For Q5 reactions, use 20-30 s per kb. For Phusion reactions, use 15-30 s per kb

StepTemp (°C)Time
I. Denature985 min.
25 - 35 cycles985-10 s
10-30 s
72
F. Extension722 min.

Information was acquired from NEB

Team:CU-Boulder - 2015.igem.org