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Source: Purified from an E. coli strain carrying a DNAPolymerase I large fragment overproducing plasmid.

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 nmoles of dNTPs to an acid insoluble form in 30 minutes at 37°C.

Reaction conditions: 50 mM Tris-HCl (pH 7.6 @ 25oC), 5 mM MgCl2, 1 mM DTT and dNTPs (not included). Klenow fragment is also 50% active in all five standard Jena Bioscience buffers when supplemented with dNTPs.

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 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 longerreaction times may result in recessed ends due to the 3′
5′ exonuclease activity of the enzyme.

Quality control: The enzyme is greater than 98% pure as indicated by SDS-polyacrylamide gel electrophoresis and contains no detected endonuclease activity. Incubation of 10 U of Klenow with supercoiled plasmid DNA produced no nicked molecules after 20 hours at 37oC as determined by agarose gel electrophoresis analysis.

The Klenow Fragment is a proteolytic product of E. coli DNA polymerase I that catalyzes the 5'→3' synthesis of DNA but has lost the 5'→3' exonuclease activity. The exonuclease-deficient variant is genetically engineered to abolish its 3'→5' exonuclease activity. The enzyme is suited for second strand synthesis, particularly for synthesis of DNA using fluorescent nucleotide analogs (1).

Standard DNA Polymerase Assay Conditions: The polymerase activity is assayed in 1x Klenow Fragment reaction buffer including 300 μM dNTPs, M13mp18 ss-DNA, and M13 universal primer.

Quantification of ds-DNA is performed using PicoGreen® (2).

Absence of contaminants: Tested for the absence of endo- and exodeoxyribonucleases.

Unit definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmoles of dNTPs into acid-insoluble material in 30 minutes at 37°C.