Klenow Fragment (3'→5' exo-)

Product Descriptions

Klenow Fragment (3'→5' exo-) is original derived as a proteolytic fragment of E. coli DNA polymerase I. It retains the DNA polymerase activity but lacks both 5'→3' and 3'→5' exonuclease activity. The recombinant Klenow Fragment (3'→5' exo-)  is expressed in an E. coli strain that carries a cloned Klenow gene with mutations which abolish the 3'→5' exonuclease activity.

Specifications & Components

Applications

• Synthesis of second strand cDNA.

• Random primer labeling.

• Labeling of 5' overhangs.

• DNA sequencing by the Sanger dideoxy method.

Unit Definition

One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides（dNTPs）into acid insoluble material in 30 minutes at 37°C.

Highlights

• 3' A-Tailing

• Probe preparation using random primers

• Second strand cDNA synthesis

• Random primer labeling

Key Performance Data

Highly efficient 3' A-Tailing

High enzyme activity of Klenow (3'→5' exo-) measured by capillary electrophoresis (CE). An NED-labeled Blunt-end dsDNA was used as a substrate for the A-tailing experiment with Klenow (3'→5' exo-) enzyme. The CE traces showed that around 80% of the labeled substrates extended one nucleotide (dA) after 30 mins incubation at 37oC.