Kinase and Anneal Oligos
This protocol is designed for kinasing oligos to be used in cloning (such as creating a polylinker). Refer to the Site-directed mutagenesis protocol for the protocol-specific kinasing reaction conditions.
- Set up kinase reaction (set up a separate reaction for each oligo):
- 50pmol/µl oligo 1.0 µl
- 10x NEB Polynucleotide Kinase Buffer 2.5 µl
- 10 mM ATP (made fresh in H2O) 2.5 µl
- T4 Polynucleotide Kinase 1.0 µl
- ddH2O (to 25 µl)
- Note: One can use the 10X NEB T4 Ligase Buffer to substiute PNK Buffer and ATP
- Incubate at 37°C for 30 minutes.
- Anneal Oligos:
- 5’ kinased oligo (from above) 3.0 µl
- 3’ kinased oligo (from above) 3.0 µl
- 10x NEB 2 Restriction enzyme buffer 1.0 µl
- ddH2O (to 10 µl)
- Incubate at 95°C for 5 minutes.
- Incubate at 70°C for 10 minutes in metal block (in heat block or in water bath).
- Remove metal block and let reactions slowly cool in the block to room temp. (usually about an hour).