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:Pfu DNA Polymerase

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.
EasyPfu DNA Polymerase 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.
Quality Control
The absence of endodeoxyribonucleases and exodeoxyribonucleases confirmed by appropriate quality tests. Functionally tested in PCR.
10X EasyPfu Buffer with 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.
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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