DNA Polymerase I, E. coli

Source

Escherichia coli

Description

• Exhibits the 5'→3' polymerase activity
• Exhibits the 5'→3' exonuclease activity, active only on duplex DNA
• Contains the 3'→5' exonuclease, primarily active on single-stranded DNA (1)
• Ultrapure recombinant enzyme

Applications

• Used to prepare radioactive probes by nick translation (2) and random priming (3)
• Useful for end-labeling of DNA molecules wih 3' and 5' protruding tails or blunt-ended

Unit Definition

One unit is the amount of enzyme required to incorporate 10 nmoles of total deoxyribonucleotide into acid-insoluble material in 30 minutes at 37°C with DNase I-activated DNA as the template primer

Storage Buffer

50 mM potassium phosphate (pH 7.0)
0.25 mM dithiothreitol
50% (v/v) glycerol

Assay Conditions

67 mM potassium phosphate (pH 7.4)
6.7 mM MgCl₂
1 mM dithiothreitol
0.033 mM of each dCTP, dGTP, dTTP and [α-³²P] dATP
4.5 µg activated DNA
Incubation is for 30 minutes at 37°C in a total reaction volume of 100 µl

Quality Control 

All preparations are assayed for contaminating endonuclease activity. Typical preparations are greater than 95% pure, as judged by SDS polyacrylamide gel electrophoresis

Storage Conditions

Store at -20ºC
Product shipped on dry ice