T4 DNA Polymerase (exo) (2000 U)

From QIAGEN, this DNA polymerase enzyme is intended for cloning, DNA labeling and recombination applications. It catalyzes the extension of a primed DNA template in the 5′→3′ direction and exhibits a powerful 3′→5′ exonuclease activity, while lacking any 5′→3′ exonuclease or strand displacement functions. The protein is produced by a recombinant E. coli strain carrying the T4 DNA Polymerase gene.

The enzyme is supplied in 100 mM KPO4, 1 mM DTT, 0.1 mM EDTA and 50% glycerol (pH 6.5 at 25°C) and is accompanied by a 10X Blue Buffer containing 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2 and 10 mM DTT (pH 7.9 at 25°C). Quality specifications include >99% purity, 5555 U/mg specific activity, functional single- and double-stranded exonuclease activities, double-stranded endonuclease at 30 U with no conversion, and E. coli DNA contamination at 30 U with <10 copies. Storage temperature is –25°C to –15°C.

T4 DNA polymerase combines a 5'→3' polymerase activity with a 3'→5' proofreading exonuclease that removes inappropriately incorporated nucleotides; unlike E. coli DNA polymerase I, it lacks 5'→3' exonuclease activity and requires an existing single-stranded DNA template primed for synthesis. Unit activity is determined by twofold serial dilution in 50 μL reactions containing calf thymus DNA, 1X Blue Buffer, 3H-dTTP and 100 μM dNTPs, incubated for 10 minutes at 37°C. Physical purity is assessed by SDS-PAGE with silver-stain detection. Exonuclease and endonuclease assays use 50 μL reactions with radiolabeled substrates or 0.5 μg of plasmid DNA incubated for 4 hours at 37°C, while E. coli contamination is evaluated by TaqMan qPCR using 5 μL replicate samples screened at the 16S rRNA locus.