Description:Recombinant Pfu DNA polymerase expressed in E.coli is thermostable with both 5’- to 3’- DNA polymerase and 3’- to 5’-exonuclease activity. Its fidelity of DNA synthesis is much higher than that of Taq DNA polymerase.
Description
Recombinant pfu DNA polymerase (rPfu DNA polymerase) is expressed in E.coli and purified to high homogeneity. This product is thermostable with both 5’- to 3’- DNA polymerase and 3’- to 5’-exonuclease activity. Thus its fidelity of DNA synthesis is much higher than that of Taq DNA polymerase. The eznyme is ideal for a variety of applications requiring high-fidelity DNA synthesis by the polymerase chain reaction (PCR). Successful PCR using pfu DNA polymerase is readily performed requiring only slight modifications from PCR protocol enclosed in this intruduction sheet..
Purity
1、≥95% by SDS-PAGE;
2、No changes of 0.5μg plasmid DNA after incubation with 10 U rPfu DNA polymerase at 74℃ for 1 hour.
Unit Definition:
One unit is defined as the amount of enzyme required to catalyze the incorporation of 10nmol of dNTPs into acid-insoluble material in 30 minutes at 75℃.
Package
1、rPfu DNA polymerase,200 U/vial,200μL,enzyme activity>1 U/μL;
2、10 x reaction buffer without Mg2+,1ml/vial;
3、0.1mol/L MgCl2 solution,0.5ml/vial
10x Reaction Buffer without Mg2+
200 mM Tris-HCl (pH 8.75);100 mM KCl;160 mM (NH4)2SO4;1% Triton X-100;1 mg/ml BSA。
PCR Protocol
1、Reaction mixture
10x Reaction Buffer without Mg2+ 5 μL
dNTP mixture (2.5mmol/L each) 4 μL
0.1mol/L MgCl2 solution 1 μL
Primer 1 (10 μmol/L) 1μL
Primer 2 (10 μmol/L) 1μL
Template DNA 10 pg~200 ng
rPfu DNA polymerase (1 U/μL) 1μL
ddH2O up to 50 μL
2、Recommended thermal cycling conditions
Step | Temperature (℃) | Time (seconds) | Numbers |
Initial denaturation | 94 | 180 | 1 |
Denaturation | 94 | 35 | 37 cycles |
Annealing | 56-65 | 15 | |
Extension | 72 | 60 | |
Final Extension | 72 | 300 | 1 |
3、PCR result analysed by 1% agarose gel electrophoresis
M 1 2 3 4 M
M: DNA Marker 5000、3000、2000、1000、750、 500、250、100bp;
1:400bp PCR band;
2:800bp PCR band;
3:1200bp PCR band;
4:1200bp PCR band (control).
Stability
It will keep activity more than one year stored at -20℃.