DNA probes can be generated from restriction fragments, PCR reactions or by annealing complementary single-stranded DNAs. If a short (oligo) probe is being used, consider end-labelling using T4 polynucleotide kinase. However, for most probes, random priming can be used to incorporate a radioactive dNTP. The steps are: isolate the DNA and clean it up. Use a kit to label the DNA, and then a spin column to remove excess unincorporated label (free dNTPs).
Purification of DNA from agarose gels
Shake to suspend "Geneclean Spin Glassmilk"; add 400 uL to a spin filter.
Add gel slice (300 mg of gel and 5 ug DNA per filter max)
Heat to 55C for 5 minutes. Invert and flick frequently.
Spin at 13,000 rpm.
Add 500 uL "Geneclean Spin New Wash" (must add EtOH first use only).
Spin 30 seconds at 13,000 rpm.
Repeat wash with 200 uL of wash.
Empty catch tube and spin one minute to dry.
Transfer to clean catch tube.
Add 10-25 uL of "Geneclean Spin Elution" buffer. Resuspend by pipetting.
Spin 30 seconds at 13,000 rpm. (If desperate, can do second elution to up yield by about 10%).
DNA labeling with Roche High Prime kit
Adjust about 50 ng of DNA to 11 uL with water.
Boil for 10 minutes and quench in EtOH/ice slurry.
Add 5 uL relatively fresh alpha-32P-dCTP
Add 4 uL High Prime Kit
Place in heat block for 10 minutes at 37C
Add 2 uL 0.2 M EDTA to stop reaction (or heat to 65C) x 10 minutes
Add another 60 uL H2O to provide sufficient volume for column.
G-50 Spin Columns
Remove column from fridge and allow it to equilibrate to room temperature.
Flick to mix the sephadex slurry.
Remove the upper cap and then the lower (in that order).
Place in a tube and spin at about 300 rpm for a few seconds to get rid of the bulk of the fluid in the tube.
Dump the collected fluid and replace column.
Spin at 1100 g (about 2300 rpm in Beckman) for 2 minutes.
Apply above reaction to the center of the column.
Spin again at 1100 g for 5 minutes.