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 37oC.
Storage Conditions: Store at –20oC.
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 37oC 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.
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