T4 DNA Polymerase

T4 DNA Polymerase catalyzes the synthesis of DNA in the 5’→ 3’ direction and requires the presence of template and primer. This enzyme has a 3’→ 5’ exonuclease activity which is much more active than that found in DNA Polymerase I (E. coli).  Unlike E. coli DNA Polymerase I, T4 DNA Polymerase does not have a 5’→ 3’ exonuclease function..

The enzyme is available in 150 and 750 unit sizes at a concentration of 3 U/µL. The enzyme is supplied with a 10× Reaction Buffer.
 

Applications

• Removal of 3’ overhangs to form blunt ends.
• Fill-in of 5’overhangs fill-in to form blunt ends.
• Single strand deletion subcloning.
• Second strand synthesis in site-directed mutagenesis.
• Probe labeling using replacement synthesis.

Product Specifications

• Storage Buffer: 100 mM KPO₄ (pH 6.5 at 25 ^oC), 1 mM DTT, 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, 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 100 µM dNTPs.

2.     Add 1 unit T4 DNA Polymerase per microgram DNA.

3.     Incubate at 12 °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.