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Reaction for generating blunt ends on dsDNA fragments with overhangs (especially good for 5' overhangs).

Notes: Klenow Fragment (aka: Large Fragment of DNA Pol I) - Dilute to 0.5 U/µl in Klenow Dilution Buffer in a labelled µ-tube, store in the -20°ree; enzyme box.

The 0.5mM dNTPs mix (all 4 dNTPs, each at 0.5mM, diluted 20X from the 10mM dNTPs) is in a µ-tube in the -20°ree; mol bio buffers&reagents box.

This protocol mainly follows the instructions on the data sheet that came with the enzyme (Gibco # 18012-021), plus some input from "Current protocols in Molecular Biology" (the Red Book) and/or "Molecular Cloning- A Laboratory Manual (Maniatis).

Fill-In Reaction: Total Volume = 30 µl.

sterile milli-Q Water: __ µl
10X React 2 buffer: 3 µl
0.5mM dNTP mix: 1 µl
DNA to be blunted: 0.5-1 µg (or 0.1-4 µg) _µl
Klenow Fragment (0.5U/µl): 1 µl

Mix gently and spin briefly.

Incubate the reaction at room temp. for 15-30 minutes.
(Gibco says room temp. for 10-15 minutes, or 20 min on ice. Maniatis says room temp. for 30 minutes. Red-Book says 30°ree; for 15 min).

Stop the reaction by doing the 'miniprep cleanup protocol' using the blue Qiagen spin column.

Gibco and Maniatis say to stop rxn by phenol/chlroform extraction. Red Book says to stop the rxn by heating to 75°ree; for 10 min or by adding 1 µl of 0.5M EDTA. Don't want to use EDTA, and I haven't yet tried heating. It should kill enzyme, but it doesn't get rid of the nucleotides.).

Shirley Reynolds

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