Klenow Fragment, DNA Polymerase (E. coli)

Klenow, the large fragment of E. coli DNA Polymerase l enzyme which retains both the polymerase and the proofreading 3'->5’ exonuclease activities of Polymerase l.

Description:

• Lacks the 5'-exonuclease activity (1).
• Ultrapure recombinant enzyme.
• Used for Sanger dideoxy sequencing (2).
• Suitable for second strand cDNA synthesis (3).
• Used for 3'-end labeling and filling in 5'-protruding sticky ends (4).

Unit Definition: One unit is the amount of enzyme required to incorporate 10 nmoles of total nucleotide into acid-insoluble form in 30 minutes at 37^oC.

Storage Conditions: Store at –20^oC.

Storage Buffer: 50 mM potassium phosphate (pH 7.0), 0.25 mM dithiothreitol and 50% (v/v) glycerol.

Assay Conditions: 67 mM potassium phosphate (pH 7.4), 6.7 mM MgCl₂, 1 mM dithiothreitol, 0.033 mM each of dCTP, dGTP, dTTP and [a-³²P]dATP and 4.5 µg activated DNA. Incubation is at 37^oC for 30 min in a reaction volume of 100 µl.

Quality Control: All preparations are assayed for contaminating endonuclease and 5'-exonuclease activities. Typical preparations are greater than 95% pure, as judged by SDS polyacrylamide gel electrophoresis.

References:

1. Joyce, C.M. and Grindley, N.D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1830-1834.
2. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467.
3. Houdebine, L.M. (1976) Nucleic Acids Res. 3, 615-630.
4. Sambrook, J., Fritsch, E.F., and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, second edition pp. 5.34, 5.40-5.43 Cold Spring Harbor Laboratory, Cold Spring Harbor.
5. Richardson, C.C., Schildkraut, C.L., Aposhian, H.V. and Kornberg, A. (1964) J. Biol. Chem. 239, 222-232.