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Protocol for Phosphorylation of 5'-termini of DNA or Oligonucleotide
(with T4 Polynucleotide Kinase)

1. Prepare the following reaction mixture:
   

   DNA or Oligonucleotide	1-20 pmol of 5'-termini 10-50 pmol
   10X reaction buffer A for T4 Polynucleotide Kinase	2 µl
   10 mM ATP	2 µl
   Water, nuclease-free	to 19 µl
   T4 Polynucleotide Kinase	1 µl (10 u)

2. Incubate at 37°C for 20 minutes.
3. Heat at 75°C for 10 minutes.
4. Separate labeled DNA from unincorporated label by gel filtration on Sephadex G-50.

Note

For estimation of pmol of DNA ends, see appendix table.

Ordering Information

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

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Updated sausio 31, 2008 14:44