Klenow enzyme (Product No. KLEN-RO) is a DNA-dependent 5′→3′ polymerase with 3′→5′ exonuclease activity. It lacks the 5′→3′ exonuclease activity of the native enzyme. The addition of mononucleotides from dNTPs to the 3′-OH terminus of DNA is catalyzed. This property is used to synthesize DNA complementary to single-stranded DNA templates.
Protocol for using Klenow
- In a 20-μL reaction, digest 0.1 to 4 μg DNA with a restriction endonuclease.
- Add 1 μL of 0.5 mM of each dNTP.
Note: It is usually not necessary to inactivate the restriction endonuclease, to change buffers, or to repurify the DNA before adding the Klenow enzyme. If you nevertheless experience problems, try to adjust the buffer conditions to the SURE/Cut incubation Buffer M (f.c. 10 mM Tris, 10 mM MgCl ¬2, 50 mM NaCl, 1 mM DTT, pH 7.5).
- Add 1 to 5 U of Klenow enzyme and incubate at 30 °C for 15 min.
- Stop the reaction by heating to 75 °C for 10 min or by adding 1 μL of 0.5 M EDTA.
For restriction fragments produced by cleavage with different endonucleases, it is possible to repair one end selectively. This is done by cleaving with enzyme 1, repairing the ends, inactivating the Klenow fragment by heat (75 °C for 10 min), and cleaving with enzyme 2.
Note: Repair of 5′ extensions is carried out by Klenow 's polymerase activity, whereas repair of 3′ extensions is carried out by 3′->5′exonuclease activity. Due to the relative inactivity of exonuclease, this method is not desirable in cases where extensive repair of overhanging 3′ ends is required. In such situations, T4 DNA polymerase is recommended.