cDNA Random Priming
For labeling cDNA probes for use for In Situ Hybridization.
High Prime DNA Labeling Kit, 50 reactions, Roche Applied Science, #11585584001 $333
dATP-alphaS35, (250 uCi vial = 20 µl) Perkin Elmer #NEG034H250UC $172
- made first Tuesday of every month
ProbeQuant G-50 micro spin columns, 50 pack, GE Healthcare, # 28-9034-08 $199
Take radioactivity out to melt (1 h if isotope in thick glass vial).
Turn on heat block to 100° C.
In 1.5 ml tube:
- 100 ng cDNA probe
- 1 µl DTT 250 mM stock solution
- ddH20 to make 8 µl
Denature at 100 C for 10 min (put cap lock on tube). Cool on ice 2-3 minutes, spin down
- 1 µl each dCTP, dGTP, dTTP (tubes 3,4,5)
- 4 µl of 5x Klenows enzyme/hexanucleotide mixture (tube 1)
- 5 µl 35S-dATP
(20 µl total)
Spin down. Leave on bench at RT for 12-24 h. (or at 37C for 1 h).
Add 30µl TE to stop reaction.
Spin down in spin column.
Count 1 µl in 10 ml scintillation fluid.
Should be 500,000 to 2,000,000 cpm/µl
If more than 1,000,000 cpm, there may be excess unincorporated nucleotides that can be removed with a second spin column run.
Use 5-10 million cpm / ml of hybridization buffer for in situ hybridization.