- Klenow fragment -large fragment of E. coli DNA polymerase I.
- Description
- Large fragment of E.coli DNA polymerase I (Klenow fragment, EC 2.7.7.7) posses 5´-3´ polymerase avtivity (45 nt/s), and 3´-5´ exonuclease (proofreading) activity.
- Unit definition
One unit of the enzyme converts 10 nmol of dNTPs to an acid-insoluble form in 30 min at 37°C.
- Reaction buffer
- B19 (10x): 100 mM Tris-HCl pH7.5, 50 mM MgCl2, 75 mM DTT.
- Reaction conditions
For fill-in reaction DNA should be present at conc. 50 mg/ml. Add 1 U Klenow, 1 mM dNTPs (end conc.), reaction buffer and incubate 30 min at 37°C. Excessive amount of the enzyme or prolonged incubation time may result in recessed ends due to the 3´-5´ exonuclease (”proofreading“) activity.
- Storage
- Store at -20°C.
- Applications
- DNA radiolabeling by the random priming-technique, by filling-in reaction or by replacing of 3´ nucleotides; fill-in protruding 5´ termini for blunt-end formation; synthesis of the second cDNA strand.
Quality control
activity, absence of contaminating exo- und endonucleases, SDS-PAGE.
References
1. Klenow, H. et al. (1971) Eur. J. Biochem. 22, 371-381.
- Catalog #
- L1.3
L1.4
Pack size
500 U
1000 U