Klenow Enzyme (DNA Polymerase I Large Fragment)

Beschreibung

Klenow Enzyme (DNA Polymerase I Large Fragment)

Source: E. coli strain carrying a plasmid with a clone copy of DNA Polymerase I large fragment
Reagents supplied with: 10x Klenow reaction buffer
Store at: -20 °C
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.
Unit definition: One unit is defined as the amount of enzyme required to convert 10 nmol of dNTPs to an acid insoluble form in 30 minutes at 37 °C.
10X Klenow Reaction Buffer: 500 mM Tris-HCl (pH 7.6 at 25 °C), 50 mM MgCl₂, 10 mM DTT
Assay conditions: 50 mM Tris-HCl (pH 7.6 at 25 °C), 5 mM MgCl₂, 1 mM DTT and dNTPs.
Unit definition: One unit is defined as the amount of enzyme required to convert 10 nmol of dNTPs to an acid insoluble form in 30 minutes at 37 °C.
Storage buffer: 0.1 M KPO₄ (pH 6.5), 1 mM DTT and 50% glycerol.
Heat inactivation: 75 °C for 20 minutes.
Protocol for blunting DNA Fragments: Reaction conditions for removal 3’ overhangs and fill-in of 3’ recessed ends (5’ overhangs) are the same. 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 room temperature (25 °C). Stop the reaction by heating to 75 °C for 20 minutes. Alternatively, the reaction can be stopped by adding EDTA to 10 mM final concentration. The Klenow enzyme is also active in Simple Restriction enzyme buffer and Taq Polymerase buffer when supplemented with dNTPs. Note: When chewing back 3’ overhangs it’s important to include dNTPS as well, because in absence of dNTPs the 3’ →5’ exonuclease activity will lead to 3’ recessed ends.

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 Fragment with supercoiled plasmid DNA produced no nicked molecules after 20 hours at 37 °C as determined by agarose gel electrophoresis analysis.

For research use only. Not for use in diagnostic procedures.