φ29 DNA Polymerase responsible for the replication of theBacillus 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 3′→ 5′ proofreading exonuclease function (3).
Source of Protein
A recombinant E. coli strain carrying the φ29 DNA Polymerase gene from bacteriophage φ29.
10 mM Tris-HCl
100 mM KCl
0.1 mM EDTA
1 mM DTT
pH 7.4 @ 25°C
B7020 (10X φ29 DNA Polymerase Reaction Buffer)
10X φ29 DNA Polymerase Buffer (B7020)
500 mM Tris-HCl
100 mM (NH4)2SO4
40 mM DTT
100 mM MgCl2
pH 7.5 @ 25°C
1 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.
|Storage Temperature||-25 to -15°C|
|Specific Activity||83,333 U/mg|
|DS Endonuclease||100 U = no conversion|
|E.coli DNA Contamination||100 U <10 copies|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
QUALITY CONTROL ANALYSIS
Unit Characterization Assay
Unit activity was measured using a 2-fold 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, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).
Protein Concentration (OD280) Measurement
A 2.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 111,230 and molecular weight of 66,713 Daltons.
SDS-Page (Physical Purity Assessment)
2.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.
Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in greater than 80% release of TCA-soluble counts.
Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test
Replicate 5 µL samples of enzyme solution were 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. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
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Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.