IsoPol™ DNA Polymerase is active at ambient temperatures.
It exhibits 5’-3’ polymerase activity and lacks 3’-5’ exonuclease activity. The 5’-3’ exonuclease domain is truncated, thus the enzyme also lacks 5’-3’ exonuclease activity. IsoPol™ DNA Polymerase has excellent processivity and strong stand-displacement activity.
In addition, it can be heat inactivated at temperatures above 50°C.
|Figure 1. Activity of IsoPol™ DNA Polymerase incubated for 10 minutes at temperatures from 10 to 50°C. The polymerase was assayed in 10 mM Tris-HCl pH 8.5, 100 mM NaCl, 5 mM MgCl2 , 700 µg/mL denatured herring sperm DNA, 33 µM dNTPs (each), 0.125 µM [3H]-dTTP, 0.8 mM DTT, 0.02 % Triton X-100.|
Unit Definition: One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C. IsoPol™ DNA Polymerase exhibits a specific activity of ~20000 U/mg.
Relative specific activity: The relative specific activity of the IsoPol™ DNA Polymerase was compared to other commercially available polymerases by using assay conditions as mentioned in the unit definition above.
|Figure 2: Relative activity of IsoPol™ DNA Polymerase compared to other commercially available polymerases at 37°C. The enzymes were assayed in 10 mM Tris-HCl pH 8.5, 100 mM NaCl, 5 mM MgCl2 , 700 µg/mL denatured herring sperm DNA, 33 µM dNTPs (each), 0.125 µM [3H]-dTTP, 0.8 mM DTT, 0.02% Triton X-100. Activity of Phi29 polymerase was close to non-detectable in this setup.|
Strand-displacement activity: IsoPol™ DNA Polymerase exhibits similar strand-displacement activity as that of Bsu DNA polymerase I at 25°C.
Optimal reaction conditions: Optimal performance is achieved in a pH 8.5 buffer supplemented with 5 mM MgCl2 and 25-100 mM salt (NaCl or KCl), at 37°C.
Storage and Stability
The enzyme is stable at -20°C for > 1 year in the supplied storage buffer. The enzyme tolerates at least five freeze-thaw cycles (-80°C) without loss of activity.
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dsDNA endonuclease activity
25 U IsoPol™ DNA Polymerase was incubated with a supercoiled plasmid (1 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any transformation of closed circular DNA to nicked DNA.
ssDNA endonuclease activity
25 U IsoPol™ DNA Polymerase was incubated with M13 ssDNA (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of ssDNA degradation.
Additional Data and Information
We are pleased to provide data and information relating to IsoPol™ DNA Polymerase. The Product is an Early Access Product, and further product information will be generated continuously. Available data includes; buffer and storage conditions, optimal conditions, activity processivity, and strand displacement data.
We appreciate suggestions for product modifications.