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Protocol for Labeling 5'-protruding Termini of DNA by Exchange Reaction
(with T4 Polynucleotide Kinase)

1. Prepare the following reaction mixture:
   

   digested DNA	1-20 pmol of 5'-termini
   10X reaction buffer B for T4 Polynucleotide Kinase	2 µl
   [gama-32P or gama-33P]-ATP	40 pmol
   24% (w/v) PEG 6000 solution	4 µl
   Water, nuclease-free	to 19 µl
   T4 Polynucleotide Kinase	1 µl (10 u)

2. Incubate at 37°C for 30 minutes.
3. Add 1 µl 0.5 M EDTA (pH 8.0) and heat at 75°C for 10 minutes.
4. Separate labeled DNA from unincorporated label by gel filtration on Sephadex G-50.

Note

• If an ethanol solution of [gama-³²P or gama-³³P]-ATP is used, dry the required amount of ATP under vacuum and dissolve in water, nuclease-free.
• The ATP concentration should be at least 1 µM in the forward reaction and at least 2 µM in the exchange reaction (1, 2).

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 3.10.2-3.10.5, 1994-2005.

Ordering Information

T4 Polynucleotide Kinase Calf Intestine Alkaline Phosphatase (CIAP) Shrimp Alkaline Phosphatase (SAP) ATP 0.5 M EDTA, pH 8.0 Water, nuclease-free DEPC-treated Water, molecular biology grade

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Updated sausio 31, 2008 14:45