Klenow (3ʹ→ 5ʹ exo–)

• OEM by QIAGEN offers bulk manufacturing of Klenow (3ʹ→ 5ʹ exo–) in custom formulations
• Derivative of E. coli DNA Polymerase I
• No 3ʹ→ 5ʹ exonuclease activity
• No 5'➞3' exonuclease activity

Klenow (3ʹ→5ʹ exo–) is a mesophilic DNA polymerase deficient in both proofreading (3ʹ→5ʹ ) and nick-translation (5ʹ→3ʹ) nuclease activities that exhibits 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.

The enzyme is supplied in 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C.

10x Blue Buffer (B0110) contains the following: 100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl₂ and 10 mM DTT; pH 7.9 at 25°C,

Properties

• Storage temperature: –25°C to –15°C   
• Molecular weight: 68,100 Daltons

Source of recombinant enzyme protein
The protein is produced by a recombinant E. coli strain carrying the Klenow (3ʹ→5ʹ exo–) gene.

Unit definition:
One 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.

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in a glycerol (50%) containing Klenow (3ʹ→5ʹ exo–) storage solution and added to 50 µl reactions containing calf thymus DNA, 1x Blue Buffer, 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 37°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 exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-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.

UDG contamination is evaluated in a 50 µl reaction containing tritiated uracil DNA and 10 µl enzyme solution incubated for 60 minutes at 37°C under standard UDG unit characterization conditions

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

• DNA labeling
• A-tailing