Klenow Fragment is the large fragment of DNA Polymerase I that retains its 5’-3’ polymerase, 3’-5’ exonuclease and strand displacement activities. The enzyme lacks the 5’-3’ exonuclease activity of intact DNA polymerase I. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5’ termini.
The enzyme is greater than 98 % pure as indicated by SDS-polyacrylamide gel electrophoresis and contains no detected endonuclease activity. Incubation of 10 units of Klenow with supercoiled plasmid DNA produced no nicked molecules after 20 hours at 37 °C as determined by agarose gel electrophoresis analysis.
- DNA blunting by filling-in 5’-overhangs with unlabeled or labeled dNTPs
- cDNA second-strand synthesis
- Generate single-stranded DNA probes using random primers
- Site-directed DNA mutagenesis using synthetic oligonucleotides
- Blunting 3’-overhangs by Dideoxy DNA sequencing of single- or double-stranded DNA templates
- Isolated from a recombinant source
- Generates probes using random primers
- Strand displacement activity
- Dideoxy sequencing compatible
- 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.
- 100 mM KP04 pH 6.5, 1mM DTT and 50% [v/v] glycerol.
- 10x Reaction Buffer: 500 mM Tris-HCI pH 7.6 at 25 °C, 50 mM mgC12 and 10 mM DTT.