Maheshri:Kinase

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):
 * 2) *50pmol/µl oligo 1.0 µl
 * 3) *10x NEB Polynucleotide Kinase Buffer 2.5 µl
 * 4) *10 mM ATP (made fresh in H2O) 2.5 µl
 * 5) *T4 Polynucleotide Kinase 1.0 µl
 * 6) *ddH2O (to 25 µl)
 * 7) *Note: One can use the 10X NEB T4 Ligase Buffer to substiute PNK Buffer and ATP
 * 8) Incubate at 37°C for 30 minutes.
 * 9) Anneal Oligos:
 * 10) *5’ kinased oligo (from above) 3.0 µl
 * 11) *3’ kinased oligo (from above) 3.0 µl
 * 12) *10x NEB 2 Restriction enzyme buffer 1.0 µl
 * 13) *ddH2O (to 10 µl)
 * 14) Incubate at 95°C for 5 minutes.
 * 15) Incubate at 70°C for 10 minutes in metal block (in heat block or in water bath).
 * 16) Remove metal block and let reactions slowly cool in the block to room temp. (usually about an hour).