Maheshri:Kinase

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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.

1. 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
2. Incubate at 37°C for 30 minutes.
3. 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)
4. Incubate at 95°C for 5 minutes.
5. Incubate at 70°C for 10 minutes in metal block (in heat block or in water bath).
6. Remove metal block and let reactions slowly cool in block to room temp. (usually about an hour).