φ29 DNA Polymerase

OEM by QIAGEN offers bulk manufacturing of φ29 DNA Polymerase in custom formulations.

S_1143_6_OEM_Generic_Product
Let’s talk custom
Need this product modified? Contact us to get started with tailored OEM solutions.
φ29 DNA Polymerase (high concentration)

Cat. No. / ID: P7020-HC-L

2,000 U (evaulation pack) of φ29 DNA Polymerase (100,000 U/ml) and 10X φ29 DNA Polymerase Reaction Buffer.
Size
φ29 DNA Polymerase (high concentration)
φ29 DNA Polymerase (low concentration)
The φ29 DNA Polymerase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Let’s talk custom
Need this product modified? Contact us to get started with tailored OEM solutions.

Features

  • Highly processive DNA polymerase
  • Powerful strand displacement activity
  • 3’→5’ exonuclease activity (proofreading)

Product Details

φ29 DNA Polymerase is responsible for the replication of the Bacillus subtilis phage φ29 (1). The enzyme is a highly processive DNA polymerase (up to 70,000 base insertions per binding event) with a powerful strand displacement activity (2) and a high fidelity 3ʹ→5ʹ proofreading exonuclease function (3).


The enzyme is supplied in 10 mM Tris-HCl, 100 mM KCl, 0,1 mM EDTA, 1 mM DTT, 0.5% Tween-20, 0.5% NP-40 and 50% glycerol; pH 7.4 at 25°C,

10X φ29 DNA Polymerase Reaction Buffer (cat. no. B7020) contains the following: 500 mM Tris-HClm 100 mM (NH4)2SO4, 40 mM DTT and 100 mM MgCl2; pH 7.5 at 25°C.

 

Ask about REACH compliant formulations.

Performance

Polymerase properties

  • Storage temperature: –25°C to –15°C
Test Units tested Specification
Purity n/a >99%
Specific activity n/a 83,333 U/mg
Single-stranded exonuclease n/a Functional
Double-stranded endonuclease 100 U No conversion
E. coli DNA contamination 100 U 83,333 U/mg

References

  1. Blanco, L., and Salas, M. (1984) Proc. Natl. Acad. Sci. 81:5325.
  2. Blanco, L., et al. (1989) J. Biol. Chem. 264:8935.
  3. Garmendia, C., et al. (1992) J. Biol. Chem. 267:2594.

Principle

Source of recombinant enzyme protein
The protein is produced by a recombinant E. coli strain carrying the φ29 DNA Polymerase gene from bacteriophage φ29.

Unit definition:
One unit is defined as the amount of polymerase required to convert 0.5 pmol of dTTP into acid insoluble material in 10 minutes at 30°C.

Molecular weight: 66,713 Daltons

Procedure

Quality control analysis
Unit activity was measured using twofold serial dilution method. Dilutions of enzyme were made in 1X φ29 DNA Polymerase reaction buffer and added to 50 µl reactions containing λ Hind III DNA, 1X φ29 DNA Polymerase Reaction Buffer, 3H-dTTP, 0.2 µM dTTP and 200 µM dATP, dCTP, dGTP. Reactions were incubated 10 minutes at 30°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

Protein concentration is determined by OD280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

 

Applications

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

  • Whole genome amplification (WGA)
  • Rolling circle amplification (RCA)
  • Multiple displacement amplification (MDA)