Klenow DNA Polymerase

Description:
Klenow (3′→ 5′ exo-) is a mesophilic DNA polymerase deficient in both proofreading (3′→ 5′) and nick-translation (5′→ 3′) nuclease activities, and that displays a moderate strand displacement activity during DNA synthesis. The protein is expressed as a truncated product of the E.coli PolA gene and contains the D355A and E357A mutations.

Specific Activity:
5,000U/mg

• Random priming labeling
• DNA sequencing by the Sanger dideoxy method
• Second strand cDNA synthesis
• Second strand synthesis in mutagenesis protocols

Supplied in:
20 mM Tris-HCl
1 mM dithiothreitol
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C

Supplied with:
10X Blue Buffer

10X Blue Buffer :
500mM 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.

Nuclease Contamination 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 less than 0.1% 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 less than 0.1% release of TCA-soluble counts.

Endonuclease Activity:
A 50 μl reaction containing 1 μg of pENZuC 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.

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 Klenow (3′→5′ exo-) storage solution ([Klenow]f = 0.12-0.002μg/μL) and added to 50 μL reactions containing 4 μg Calf Thymus DNA, 1X Blue 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 55,450 and molecular weight of 68,067 Daltons. Acceptance for this assay is +/- 5% of reference sample.

Recommended Storage Conditions:
-20°C