PNK Treatment of DNA Ends: Difference between revisions
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*The above website outlines a protocol for use that is modified and summarized below. | *The above website outlines a protocol for use that is modified and summarized below. | ||
*10 | *10 μL Reaction Mix | ||
**1 | **1 μL PNK stock (10,000 U/ml) | ||
**1 | **1 μL T4 Ligase Buffer | ||
**8 | **8 μL Substrate | ||
*Reaction Conditions | *Reaction Conditions | ||
*37 | *37°C for 30mins | ||
*65 | *65°C for 20mins | ||
*Store at 4 | *Store at 4°C | ||
*The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although the author has not done this. | *The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although the author has not done this. | ||
Revision as of 16:07, 12 May 2005
This protocol is used to add a phosphate group to the 5' end of a DNA molecule. Most primers for example are ordered without this being added as it requires an extra synthesis step and hence greater cost. However subsequent ligation steps are more efficient if these phosphate groups are added.
- T4 Polynucleotide Kinase is an enzyme that can perform this on blunt or overhanging DNA ends.
- The above website outlines a protocol for use that is modified and summarized below.
- 10 μL Reaction Mix
- 1 μL PNK stock (10,000 U/ml)
- 1 μL T4 Ligase Buffer
- 8 μL Substrate
- Reaction Conditions
- 37°C for 30mins
- 65°C for 20mins
- Store at 4°C
- The T4 Ligase Buffer provides the required ATP and substitutes for the PNK Buffer and ATP in the NEB protocol. It is actually possible to perform the PNK step and a ligation step simultaneously although the author has not done this.
