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Team:British Columbia/Notebook/Protocols/T4PNK
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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.) | ||
Latest revision as of 01:13, 23 September 2013
iGEM Home
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.)
