PCR

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Protocol for Phusion

  • Add the following volumes to a round-lidded PCR tube:
Reagents
Volume (µL)
ddH2O
up to 50 µL
HF 5x buffer
10
ddNTPs
1
Primer A
0.5
Primer B
0.5
DNA
X
DNA polymerase
0.5-2
DMSO (optional)
(1.5)
Total
50

Note: GC buffer can be used instead of HF buffer when using GC-rich templates

  • Add the tube to the thermocycler and use the following times:
Step Temperature (°C) Time (s) Number of cycles

Initial denaturation

98 30 1
Denaturation 98 5-10 35-45
Annealing

>20nt; Tm + 3°C

<20nt; Tm (lower of the two primers)

10-30

(time depends on complexity)

35-45
Extension 72 15-30/kb 35-45
Final extension 72 5 min 1
Infinite hold 4 hold 1


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