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.
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