Description
Klenow Fragment is a mesophilic DNA polymerase derived from the E. coli Polymerase I DNA-dependent repair enzyme. The enzyme exhibits DNA synthesis and proofreading (3ʹ→5ʹ) nuclease activities and, in the absence of the holoenzyme’s (5ʹ→3ʹ) nuclease domain, displays a moderate strand displacement activity during DNA synthesis. The protein is expressed as a truncated E. coli PolA gene product. The enzyme is supplied in 100 mM KPO4, 1.0 mM DTT, 0.1mM EDTA and 50% glycerol; pH 7.4 at 25°C. The buffer solution known as 10x Blue Buffer has a pH of 7.9 at 25°C and includes 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl 2 and 10 mM DTT.
Storage temperature: –25°C to –15°C Molecular weight: 68,202 Daltons Test Specification Purity >99% Specific activity 5000 U/mg Single-stranded exonuclease 50 U; functional Double-stranded exonuclease 50 U; functional Double-stranded endonuclease 50 U; no conversion E. coli DNA contamination 50 U; <10 copies
The protein is produced by a recombinant E. coli strain carrying the Klenow Fragment gene. 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.
Usage Instructions 1. Set up the following reaction mixture: Dissolve DNA in 1x Blue Buffer (B0110) Add dNTPs (each at a final concentration of 33 µM) Add 1 U of Klenow Fragment per microgram of DNA 2. Incubate the reaction mixture at 25°C for 15 minutes. 3. Stop the reaction by adding EDTA (final concentration of 10mM) and heating at 75°C for 20 minutes. Quality Control Unit activity was measured using a two-fold serial dilution method. Dilutions of the enzyme were made in a 50% glycerol Klenow (3ʹ→5ʹ exo–) storage solution and added to 50 µL reactions containing calf thymus DNA, 1x Klenow Reaction Buffer, 3H-dTTP and 100 µM dNTPs. Reactions were incubated for 10 minutes at 37°C, placed on ice and analyzed using the method of Sambrook and Russel (Molecular Cloning, v3, 2001, pp. A8.25- A8.26) Protein concentration is determined by OD 280 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 band's mass 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.
