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Protocol for Labeling Recessed 3'-termini of Double-stranded DNA
(with Klenow Fragment)

1. Prepare the following reaction mixture:
   

   Digested DNA (aqueous solution)	10-15 µl (0.1-4 µg)
   10X reaction buffer for Klenow Fragment	2 µl
   [alfa-32P]-dNTP, ~15-30 TBq/mmol (400-800 Ci/mmol) or [alfa-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	0.74 MBq (20 µCi) 2.96 MBq (80 µCi)
   3 dNTP Mix, 2 mM each (without a labeled dNTP)	2.5 µl (0.25 mM final concentration)
   Klenow fragment	0.1 µl (1 u)
   Water, nuclease-free	to 20 µl

2. Incubate the mixture at 30°C for 15 minutes.
3. Stop the reaction by heating at 75°C for 10 minutes.

Reference

1. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.5.7-3.5.10, 1994-2005.

Ordering Information

Klenow Fragment dNTP Mixes dNTP Set Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ H Minus M-MuLV Reverse Transcriptase RNase H, E.coli Pyrophosphatase, Inorganic (from yeast) 0.5 M EDTA, pH 8.0 Water, nuclease-free

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Updated sausio 31, 2008 15:12