Promega, M2051, DNA Polymerase I

DNA Polymerase I catalyzes the template-directed polymerization of nucleotides into duplex DNA in a 5´→3´ direction. DNA Polymerase I possesses a 3´→5´ exonuclease activity or "proofreading" function, which lowers the error rate during DNA replication, and also contains a 5´→3´ exonuclease activity, which enables the enzyme to replace nucleotides in the growing strand of DNA by nick translation. The enzyme, purified from recombinant E. coli , is capable of catalyzing de novo synthesis of synthetic homopolymers and provides a convenient method for the preparation of a variety of defined DNA substrates. DNA Pol I is provided with 10X Reaction Buffer: 500mM Tris-HCl (pH 7.2 at 25°C), 100mM MgSO 4 , 1mM DTT.
Applications
Labeling of DNA to high specific radioactivity by nick translation.
Second-strand cDNA synthesis.
References
Kelly, R.B. et al. (1970) J. Biol. Chem. 245 , 39–45.
Harwood, S.J. et al. (1970) J. Biol. Chem. 245 , 5614–24.
Storage Buffer: 50mM Tris-HCl (pH 7.5 at 25°C), 1mM DTT, 0.1mM EDTA, 50% (v/v) glycerol.
Source: Recombinant E. coli strain.
QC Tests: Activity, SDS-PAGE/purity, endonuclease.
Unit Definition: One unit is defined as the amount of enzyme that incorporates 10nmol of deoxyribonucleotides into TCA-insoluble material in 30 minutes at 37°C. The reaction conditions are: 67mM potassium phosphate (pH 7.4 at 25°C), 6.7mM MgCl 2 , 1mM DTT, 50μg/ml activated calf thymus DNA and 33μM dATP, dCTP, dGTP and dTTP (a mix of unlabeled and [ 3 H]dTTP).