Description
DNA Polymerase I is a mesophilic DNA polymerase that exhibits 5’-3’ DNA synthesis, in addition to both 3′→5′ and 5′→3′ exonuclease activities. The combination of DNA synthesis and 5′→3′ nuclease characteristics enable nick-translation during DNA synthesis. The protein is produced by a recombinant E. coli strain carrying the PolA gene. The enzyme is supplied in 25 mM Tris-HCl 0.1 mM EDTA 1.0 mM dithiothreitol 50% glycerol pH 7.4 at 25°C. It is supplied with a 10X Blue Buffer (B0110) containing: 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl 2, 10 mM DTT, pH 7.9 at 25°C.
Assay Specification Purity >99% Specific activity 6850 U/mg Single-stranded exonuclease Functional Double-stranded exonuclease Functional Double-stranded endonuclease 200 U; no conversion E. coli DNA contamination 200 U; <10 copies
DNA Polymerase I is a DNA-dependent DNA polymerase with inherent 3´→ 5´ and 5´→ 3´ exonuclease activities. The 5´→ 3´ exonuclease activity removes nucleotides ahead of the growing DNA chain, allowing nick-translation.
Instructions for using DNA Polymerase I are provided in the corresponding kit protocol in the resources below. Quality Control Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X Blue Reaction Buffer and added to 50 µL reactions containing Calf Thymus DNA, 1X Blue Reaction Buffer, 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using a TCA-precipitation method. Protein concentration was determined by OD 280 absorbance. Physical purity was evaluated by SDS-PAGE by loading a 2.0 μL of concentrated enzyme solution on a denaturing 4-20% Tris-Glycine gel flanked by a broad-range MW marker and 2.0 μL of a 1:100 dilution of the sample. Single-stranded exonuclease was determined in 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. Double-stranded exonuclease activity was determined in 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. Double-stranded endonuclease activity was determined in a 50 μL reaction containing 1 μg of pBR322 DNA and 10 μL of enzyme solution incubated for 4 hours at 37°C. E.coli contamination was evaluated using 5 μL samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus.
