University of Virginia Health System

We use a Random priming kit from Roche Applied Science

1. DNA fragment in low melt agarose
re-boil DNA 3'

cool to 37°C, 1-2', 15' max

2. mix in order:

3 µl dATP, dGTP, dTTP mixture 1:1:1 (solutions 2,4,5)
2 µl reaction mix (soln. 6)
9 µl DNA fragment at 37°C in low melt agarose (~0.7% or less is best)

behind plexiglass shield add: 5 µl [alpha-32P]-dCTP, 3000 Ci/mmole, aqueous
transfer to ice, add: 1 µl Klenow enzyme (soln.7)
20 µl total

incubate 30' at 37?C

3. to stop reaction, add 50µl 10 mM Tris pH 8.0, 30 mM EDTA with 50 µg/ml tRNA (="STOP SOLUTION" kept at 4°C)

4. Push-column purify incorporated, radioactive DNA (Stratagene push-column, or use spin-column).

5. measure volume in µl
6. count 1 µl in TE (Cerenkov radiation)
multiply cpm/µl by µl volume measured to calculate total cpm
7. boil 5' to denature for hybridization, quench on ice, add to blot(s)