cDNA Random Priming

From MagnetoWiki
Jump to: navigation, search

For labeling cDNA probes for use for In Situ Hybridization.

Reagents:

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

Procedure:

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

Add:

  • 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.