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 that protocol-specific kinasing reaction conditions.

1. Set up kinase reaction (set up a separtate reaction for ewach 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 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. Incubalte at 70°C for 10 minutes in (metal block) water bath
6. Remove metal block and let reactions slow cool to room temp. (usually about an hour).