DNA Polymerase I

Description:
DNA Polymerase I is a mesophilic DNA polymerase that exhibits 5′-3′ DNA synthesis in addition to both 3′→ 5′ and 5′→ 3′ exonuclease activities. The combination of DNA synthesis and 5′→ 3′ nuclease characteristics enable nick-translation during DNA synthesis.

Applications:

• Nick translation of DNA to obtain probes with a high specific activity
• Second strand synthesis of cDNA

Source:
A recombinant E. coli strain carrying the PolA gene.

Specific Activity:
6,850U/mg

Supplied in:
25 mM Tris-HCl
0.1 mM EDTA
1.0 mM dithiothreitol
50% glycerol
pH 7.4 @ 25°C

Supplied with:
10X Blue Buffer

10X Blue Buffer
500 mM NaCl
100 mM Tris-HCl
100 mM MgCl2
10 mM DTT
pH 7.9 @ 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 37°C.

Functional Tests

Single-Stranded Exonuclease Activity:
A 50 µL reaction containing 15,000 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 Exonuclease Activity:
A 50 µL reaction containing 15,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in greater than 50% release of TCA-soluble counts.

Nuclease Contamination Tests

Endonuclease Activity:
A 50 µL reaction containing 1 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 heat denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus. Based on no template control Ct values, the detection limit of this assay is <10 copies genome/sample.

Quality Control Analysis

Unit Characterization Assay:
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in a glycerol (50%) containing DNA Polymease I storage solution ([Pol I]f = 0.12-0.002µg/µL) and added to 50 µL reactions containing
4 µg Calf Thymus DNA, 1X Blue Reaction Buffer, 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).

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.

Protein Concentration (OD280) Measurement:
A 3.0 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 80,790 and molecular weight of 103,120 Daltons. Acceptance for this assay is +/- 5% of reference sample.

Recommended Storage Condition:
-20°C