PCR using Pfu Polymerase

6/19/2002 Juli Grose



Description

PfuTurbo DNA Polymerase (Stratagene catalog #600250) is an "enhanced" version of Pfu DNA polymerase for high-fidelity PCR (for details see the Stratagene instruction manual). Pfu has significantly lower error rates (in part due to its proofreading ability).

Use Pfu as described below and only if NECESSARY (ie when cloning). Efficient sequencing may be done by running 100 uL reaction with TAQ in 5 separate capillary tubes (thus reducing the probability of an abundant product with a bp change due to PCR).

Keep Pfu on ice...it is EXPENSIVE.

Protocol

1. Standard reaction mixture (scale down for trial reactions)

10 uL DMSO (stock is located in the kitchen)
10 uL mPfuB buffer (supplied by Eric)
10 uL NTPs
10 uL each primer (5 uM each)
10 uL template DNA (100 ng/ul)
2 uL PfuTurbo DNA polymerase (5U)
48 uL sterile dH20

100 uL total reaction volume

2. Standard PCR cycling program (Idaho Tech AirCycler)

Hold: 2' at 95°C

35 Cycles:
15" at 94°C
15" at annealing temp. (Tm-5°C)
3' per kb at 72°C

Hold: 10' at 72°C

Note: The fifteen second holds during cycling are essential for efficient PCR

Any Questions? Ask Juli!


Last Update: Thursday, 19-Jun-2014 11:47:22 PDT
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Eric Kofoid eckofoid at ucdavis.edu