Description
T4 DNA Polymerase catalyzes the extension of a primed DNA template in the 5′→3′ direction. This enzyme 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 KPO4,1mM DTT, 0.1mM EDTA, 50% glycerol pH 6.5 at 25⁰C. It is supplied with a 10X Blue Buffer (B0110) containing: 500mM NaCl, 100mM Tris-HCl, 100mM MgCl 2, 10mM DTT, pH 7.9 at 25⁰C.
Assay Specification Purity >99% Specific activity 5555 U/mg Single-stranded exonuclease Functional Double-stranded exonuclease Functional Double-stranded endonuclease 30 U; no conversion E. coli DNA contamination 30 U; <10 copies
T4 DNA polymerase has two enzymatic activities: a polymerase activity, which catalyzes DNA synthesis from the 5' to 3' end, and a 3'→5' exonuclease activity, the so-called proofreading function, which makes it possible to recognize the incorporation of an inappropriate nucleotide and then remove it from the DNA again. Unlike E. coli DNA polymerase I, it lacks the 5'→3' exonuclease activity. The T4 DNA polymerase always uses an already existing single strand of DNA as a template and requires a primer for the synthesis of a new, complementary strand.
Instructions for using T4 DNA Polymerase are provided in the corresponding kit protocol in the resources below. Quality Control Unit activity was measured using a twofold serial dilution method. Dilutions of the enzyme were made in 1X reaction buffer and added to 50 μL reactions containing calf thymus DNA, 1X Blue Buffer, 3H-dTTP and 100 μM dNTPs. Reactions were incubated for 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). Protein concentration was determined by OD 280 absorbance. Physical purity was 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 protein of interest band in the diluted sample. Single-stranded exonuclease was 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 was 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 was 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 was evaluated using 5 μL replicate samples of enzyme solution 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.
