Kinase Reaction for Oligonucleotides

1. Set up reactions:
   2 µL oligo (300 ng)
   2 µL 10X Kinase Buffer
   2 µL gamma 32P-dATP (450 µCi)
   1 µL polynucleotide kinase (PNK)
   12 µL H2O
2. Incubate at 37ºC for 60 minutes.
3. Use spin column to remove unincorporated nucleotides:
   Increase reaction volume to 100 µL
   Add 1 µL of blue dye and 1 µL red dye.
   Run over spin column:
   • Start with empty 1 mL syringe.
   • Add cotton plug and tamp down with plunger.
   • Fill with column material. (Use either G25-SephaDex or P5.)
   • Place filled syringe in 15 mL Falcon tube.
   • Spin down 2 minutes in table top clinical.
   • Discard flow-thru. Repeat until column material is almost to top of syringe.
   • Place column in new 15 mL Falcon tube.
   • Load reaction (with dyes) onto column.
   • Spin down 2 minutes in table top clinical.
   • Collect flow-thru. Count 1 µL in scintilation counter.
   • Add remainder of probe to 50 mL pre-hybridization buffer.
   • Store probes at -20ºC when not in use.