| DNA Polymerase I Large Fragment (Klenow Fragment) |
 Technical Brochure |
Description: The Klenow Fragment lacks the 5′→3′ exonuclease activity of intact DNA Polymerase I but retains the 5′→3′ polymerase, the 3′→5′ exonuclease and the strand displacement activities. Source: Purified from an E. coli strain carrying a DNA Polymerase I large fragment overproducing plasmid. 1X Klenow Reaction Buffer: Reaction conditions: 50 mM Tris-HCl (pH 7.6 @ 25oC), 5 mM MgCl2, 1 mM DTT and dNTPs. Klenow fragment is also 50% active in all five standard MINOTECH buffers when supplemented with dNTPs. Unit definition: One unit is defined as the amount of enzyme required to convert 10 nmoles of dNTPs to an acid insoluble form in 30 minutes at 37oC. Quality control: The enzyme is greater than 98% pure as indicated by SDS-polyacrylamide gel electrophoresis and contains no detected endonuclease activity. Incubation of 10U of Klenow with supercoiled plasmid DNA produced no nicked molecules after 20 hours at 37oC as determined by agarose gel electrophoresis analysis. |
Storage buffer: 0.1 M KPO4 (pH 6.5), 1 mM DTT and 50% glycerol. Store at -20oC.
Heat inactivation: 75oC for 20 minutes. Fill-in conditions: Dissolve 0.1-4 μg of digested DNA in 1x Klenow reaction buffer supplemented with 40 μM each dNTP. Add 1 unit Klenow per μg DNA and incubate 15 minutes at 25oC. Stop the reaction by adding EDTA to 10 mM final concentration and heating at 75oC for 10 minutes. Note: excessive amounts of enzyme or longer reaction times may result in recessed ends due to the 3′→5′ exonuclease activity of the enzyme. Reagents supplied: 10x Klenow Reaction buffer |
