DNA Polymerase I, Large (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.

Klenow Fragment is also active in any restriction enzyme reaction buffer and T4 DNA Ligase reaction buffer when supplemented with dNTPs.

Source: E. coli strain harboring the plasmid that directs the synthesis of Klenow fragment

Reaction buffer:

• 10 mM Tris-HCl (pH 7.5)

• 5 mM MgCl₂

• 5 mM dithiothreitol

Quality Assurance: Purified free of contaminating endonucleases and exonucleases

Unit Definition: One unit of the enzyme catalyzes the incorporation of 0.5 pmol of dCMP into a polynucleotide fraction (absorbed on DE-81) in 10 min at 30°C

Concentration: 5 – 50 units/μl

Applications:

• DNA sequencing by the Sanger dideoxy method

• Fill-in of 5´-overhangs to form blunt-ends

• Removal of 3´-overhangs to form blunt-ends

• Second strand cDNA synthesis

• Second strand synthesis in mutagenesis protocols

Storage conditions: -20°C

Storage buffer:

• 100 mM KPO₄ (pH 6.5)

• 1 mM dithiothreitol

• 50% glycerol