Klenow Fragment

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Reagents Supplied

10X DNA Polymerase I (Klenow) Reaction Buffer

Source

Escherichia coli

Description

• Lacks 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

Heat Inactivation

75ºC for 15 minutes

Concentration

5 - 10 units/µl

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 for 30 minutes at 37ºC in a total 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

Storage Conditions

Store at -20°C
Product shipped on dry ice

References

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