Description
Klenow Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3' >5' exonuclease activity, but has lost 5' >3' exonuclease activity. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.
Source
Bacillus Subtilis in-treated DNA polymerase I
Applications
- Nick translation
- Second strand cDNA synthesis
Heat inactivation
10 minutes at 75 °C
Notes
A reaction at 25 °C during 15 minutes with 1 U/µg DNA is advised since higher amounts of enzyme or longer reaction times may cause recessed ends due to 3’ > 5’ exonuclease activity.
Unit Definition
One unit catalyses the incorporation of 10nmol of total deoxynucleotide into acid insoluble product in 30 minutes at 37°C using poly(dA-dT)-poly(dA-dT) as substrate
The Klenow fragment lacks the 5’ > 3’ exonuclease activity. It is recommended for filling in recessed 3’ ends, for second strand cDNA synthesis and for probe synthesis by random priming.