| Description | 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 (nick-translation) activity, and proofreading (3′→ 5′) exonuclease activity due to two amino acids mutations (D355A, E357A). |
| Applications | - Random-primed DNA labeling
- DNA sequencing by the Sanger dideoxy method
- Second strand cDNA synthesis
- Labeling by fill-in 5\'-overhangs of dsDNA
- Strand displacement amplification (SDA)
|
| Source | A recombinant E. coli strain carrying the mutated Klenow Fragment of E coli PolA gene. |
| 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. |
| Components | - Klenow Fragment exo-: 5,000 U/ml in 25 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25°C
- 10X Reaction Buffer: 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT, pH 7.9 @ 25°C
|
| Quality Control | - The absence of endo- and exodeoxyribonucleases confirmed by appropriate quality tests.
- Functionally tested in random-primed DNA labeling.
|
| Storage Condition | -20 °C |
