Pfu DNA Polymerase is purified from E. coli. expressing a cloned Pyrococcus furiosus DNA polymerase gene. This polymerase possesses a proofreading 3' to 5'. exonuclease activity that provides higher fidelity than Taq. Pfu DNA Polymerase makes it ideal for demanding PCR applications such as site-directed mutagenesis and PCR expression cloning.

Conc. 5 U/μl

Store at -20°C

Unit Definition

One unit of EasyPfu DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-insoluble material in 30 min at 74°C.

Storage Buffer

50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, stabilizers, and 50% (v/v) glycerol.

10X EasyPfu Buffer

20mM MgSO4 200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH4)2SO4, 100mM KCl, 1% (v/v) Triton X-100, 1mg/ml BSA and 20mM MgSO4.

Quality Control

The absence of endodeoxyribonucleases and exodeoxyribonucleases was confirmed by appropriate quality tests. Functionally tested in PCR.

Reaction Mixture Set Up

Component Volume Final Concentration
Template DNA <0.5 μg as required
Forward Primer (10 μM) 1 μl 0.2–0.4 μM each
Reverse Primer (10 μM) 1 μl 0.2–0.4 μM each
10×Buffer 5 μl 1×
2.5 mM dNTPs 4 μl 0.2 mM
EasyPfu DNA polymerase 0.5 μl 2.5 unit
ddH2O to final volume 50 μl Not applicable
Recommended thermal cycling conditions
94℃ 2-5 min
94℃ 30 sec
50-60℃ 30 sec 30-35 cycles
72℃ 2 min/1 kb
72℃ 5-10 min

Notes: MgSO4 is included in the 10×PCR Buffer at a final concentration of 2 mM, which is sufficient for most targets. For some targets, more Mg2+ may be required; use 100 mM MgSO4 prepare a titration from 2 mM to 4 mM (final concentration) in 0.25-mM increments.

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