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 MgCl ₂
• 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 KPO ₄ (pH 6.5)
• 1 mM dithiothreitol
• 50% glycerol