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Enzymatic 5' DNA Phosphorylation

Assemble the following reaction: 1 x T4 PNK Buffer, 1 mM ATP, 0.1 % Tween-20, 300 pmol of 5' termini, 0.2 U/µL of T4 Polynucleotide Kinase. Incubate the reaction at 37 °C for 1 hour, followed by heat inactivation at 65 °C for 20 minutes.

Example, for a 25 µL Reaction:

Reagent	Amount (µL)
10x T4 PNK Buffer	2.5
10 mM ATP	2.5
1 % Tween-20	2.5
Primer (100 µM)	1.5
Water	15.5
T4 PNK (10 U/µL)	0.5
Total	25

Reaction products are at 6 µM and do not need purification prior to most downstream uses (PCR, multisite mutagenesis, ligation, etc.)