Team:British Columbia/Notebook/Protocols/T4PNK

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! scope="row"|T4 PNK (10 U/µL)
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Revision as of 00:59, 23 September 2013

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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
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