Klenow Fragment (3’→5′ exo-)

Klenow Fragment (3'→5' exo-) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5´→ 3´ exonuclease activity and has mutations (D355A, E357A) which abolish the 3´→ 5´ exonuclease activity

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

• Random priming labeling.
• 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.

A-Tailing Protocol with Klenow Fragment (3'→5' exo-)

1.  Assemble the following reaction in a microcentrifuge tube on ice:

    10× Reaction Buffer                           5 µL
    10 mM dATP                                      0.5 µL
    Blunt-ended DNA                              1-5 µg
    Nuclease-free water                          to 47 µL
    Klenow Fragment (3'→5' exo-)          3 µL
    Total volume                                      50 µL

2.  Mix gently and spin down for a few seconds.

3.  Incubate at 37 °C for 30 min.

4.  Purify DNA sample on one spin column.