Klenow Fragment (Escherichia coli)

Klenow Fragment (Escherichia coli)

Source: Escherichia coli

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°C.

Storage Conditions: Store at –20°C.

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 [ α-³²P]dATP and 4.5 µg activated DNA. Incubation is at 37°C 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.

Klenow Fragment (exo-) (Escherichia coli)

Source: Escherichia coli

Large fragment of E. coli DNA Polymerase l, which lacks both the 3'->5’ exonuclease and the 5'->3’ exonuclease activities of Polymerase l.

Description:

• Devoided of both the 3'->5’ exonuclease and the 5'->3’ exonuclease activities.

• 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 nucleotides into acid-insoluble material in 30 min at 37°C.

Storage Conditions: Store at –20°C.

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 dCTP, dGTP, dTTP and [ α-³²P]dATP and 4.5 µg activated DNA. Incubation is at 37°C for 30 min in a reaction volume of 100 µl.

Quality Control: All preparations are assayed for contaminating 3'- and 5'-exonuclease and endonuclease activities.

References:

1. Derbyshire, V., Freemont, P.S., Sanderson, M.R., Beese, L., Friedman, J.M., Joyce, C.M. and Steitz, T.A. (1988) Science 240, 199-201.

2. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-546.7

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.

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