Bst DNA Polymerase (large fragment)

Description:
Bst DNA Polymerase (large fragment) is the portion of the Bacillus stearothermophilus DNA Polymerase protein that contains the 5´ ’ 3´ polymerase activity, but lacks the 5´ ’3´ exonuclease domain.

Applications:

• Isolated from a recombinant source
• Specific Activity: 120,000 units/mg
• Sequencing through problematic secondary structures

Source:
A recombinant E. coli strain carrying the BST DNA Polymerase gene (large fragment).

Supplied in:
10 mM Tris-HCl
50 mM KCl
1.0 mM dithiothreitol
0.1 mM EDTA
0.1% Triton X-100
50% glycerol
pH 7.5 @ 25°C

Supplied with:
10X PCR Buffer II

10X PCR Buffer ll
200 mM Tris-HCl
100 mM Ammonium Sulfate
100 mM KCl
20 mM MgSO4
1.0% Triton X-100
pH 8.8 @ 25°C

Unit Definition:
1 unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid insoluble material in 30 minutes at 65°C.

Nuclease Contamination Tests:

Single-Stranded Exonuclease Activity:
A 50 µL reaction containing 11,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 5.0% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity:
A 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.5% release of TCA-soluble counts.

Endonuclease Activity:
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 5 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.

Quality Control Analysis:

Unit Characterization Assay:
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme batch were made in 1X reaction buffer ([Manta]f = 0.1-0.00075 µg/µL) and added to 50 µL reactions containing 10 µg Calf Thymus DNA, 1X PCR Buffer II, 4mCi/mL 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 37°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 52,770 and molecular weight of 66,215 Daltons. Acceptance for this assay is +/- 5% of reference sample.

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.

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.

Notes:
Specific activity measured under above conditions is approximately 3X higher than cited by Beese et al. when using activated calf thymus DNA as a substrate.

References:

1. Kiefer, et al. Structure 15 January 1997. 5, 95-108.