DESCRIPTION
Klenow Fragment is the Large Fragment of DNA Polymerase I. It is a DNA-dependent polymerase with 3'-5' exonuclease activity. It lacks 5'-3' exonuclease activity of the native enzyme and is suited for random-primed labelling of DNA with random oligonucleotides and for filling-in of 5' protruding ends to blunt-ends. This enzyme can incorporate modified nucleotides (e.g., Cy3-, Cy5-, aminoallyl-, biotin-, digoxigenin- and fluorescently-labeled nucleotides) and is active in restriction enzyme, PCR, RT and T4 DNA Ligase buffers.

CONCENTRATION
5 units/µl

APPLICATION
DNA blunting by fill-in 5'-overhangs
Random-primed DNA labeling
Labeling by fill-in 5'-overhangs of dsDNA
DNA sequencing by the Sanger method
Site-specific mutagenesis of DNA with synthetic oligonucleotides
Second strand synthesis of cDNA

UNIT DEFINITION
One unit is the amount of enzyme that incorporates 10 nmoles of deoxynucleotides into acid-insoluble form in 30 minutes at 37°C.

STORAGE BUFFER
20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1 mg/ml BSA; 50% glycerol (v/v)

STORAGE TEMPERATURE
Store Klenow Enzyme below 0ºC, preferably at -20ºC, in a constant temperature freezer.

10X REACTION BUFER
100 mM Tris-HCl pH 7.5, 100 mM MgCl₂ , 10 mM DTE (= buffer L for restriction endonucleases)