Team:British Columbia/Notebook/Protocols/T4PNK

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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.
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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.  
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Incubate the reaction at 37 °C for 1 hour, followed by heat inactivation at 65 °C for 20 minutes.
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Example, for a 25 µL Reaction:
Example, for a 25 µL Reaction:
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Reaction products are at 6 µM and do not need purification prior to most downstream uses (PCR, multisite mutagenesis, ligation, etc.)
Reaction products are at 6 µM and do not need purification prior to most downstream uses (PCR, multisite mutagenesis, ligation, etc.)
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Latest revision as of 02:01, 18 October 2014

2014 UBC iGEM

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.)


Retrieved from "http://2014.igem.org/Team:British_Columbia/Notebook/Protocols/T4PNK"