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Nick translation of dsDNA with biotinylated or digoxigenin dUTP

For the use with FISH (fluorescent in situ hybridization) we always label entire plasmid DNA and do not cut out the insert.

1. mix together
2. mix carefully with finger and put for 2 hrs at 15C (cryostat)

for YAC and genomic DNA, check fragment length on 1.2 % agarose gel.
The fragment length should be 300-400 bp for YAC
and 600-1500 bp for genomic DNA (used in a CGH-experiment).
If the fragment length is larger add more DNase I.

3. stop the reaction with 5 l 0.5 mM EDTA (pH 7.4)

4. add to each tube:

2.5 l salmon sperm DNA (stock 10 mg/ml)
2.5 l yeast RNA
5.5 l 3 M NA-acetate (pH 5.6)
137.5 l icecold 100 % EtOH

6. this mixture overnight or 1 hr at -70C (precipitation)

7. centrifuge 30 min at 14000 rpm at 4C

8. remove supernatans (vacuumpomp) and dissolve in appropiate volume :

-repetitive probes in 100 l 60 % form/SSCP
-unique sequences in 50 l 50 % form/SSCP
-cosmids, YACS in 50 l TE
-libraries in 100 l TE