Klenow Fragment

Klenow Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3'→ 5' exonuclease activity, but has lost 5'→ 3' exonuclease activity. Klenow Fragment retains the polymerization fidelity of the holoenzyme without degrading 5' termini.

The enzyme is available in 200 and 1000 unit sizes at a concentration of 5 U/µL. The enzyme is supplied with a 10× Reaction Buffer.

Applications

• Fill-in of 5´ overhangs to form blunt ends.
• Removal of 3´ overhangs to form blunt ends.
• DNA sequencing by the Sanger dideoxy method.
• Second strand cDNA synthesis.
• Second strand synthesis in mutagenesis protocols.

Product Specifications

• Storage Buffer: 25 mM Tris-HCl (pH 7.4 at 25 ^oC), 1 mM DTT, 0.1 mM EDTA, and 50% (v/v) glycerol.
• Unit Definition: One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.
• Unit Assay Conditions: 1X Reaction Buffer, 33 µM dNTPs including [³H]-dTTP and 70 µg/ml denatured herring sperm DNA.

Protocol for blunting ends by 3’ overhang removal and 5’ overhang end fill-in

1. Dissolve DNA sample in 1X reaction buffer supplemented with 33 µM dNTPs.

2. Add 1 unit Klenow Fragment per microgram DNA.

3. Incubate at 25 °C for 15 min.

4. Stop the reaction by heating at 75 °C for 20 min or adding EDTA to a final concentration of 10 mM.