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

• x µl bidestilized water till endvolume is 50 ml
  
• 5 µl 10xnick translationbuffer
  
• 5 µl nucleotide mix (endconcentration = 2.5 mM):
  • for each nucleotide (dATP,dGTP and dCTP) (stock concentration = 100 mM):
    
  • 3 µl stock solution + 27 µl bidest (1)
    
  • 25 µl of each (1) +25 µl H20 = 100 µl mix
    
• 2.5 µl bio-16-dUTP (0.4 mM, ready for use)
  
• 5 µl 100 mM DTT
  
• x µl DNA (usual 1 mg disolved in 1 ml TE)
  
• 5 µl DNase I (stock 1 mg/ml, dilute 1/1000 in H20, prepare fresh every time)
  
• x µl DNA polymerase (endconcentration must be = 30 U)
  
  

2. mix carefully with finger and put for 2 hrs at 15°C (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 µl 0.5 mM EDTA (pH 7.4)

4. add to each tube:

2.5 µl salmon sperm DNA (stock 10 mg/ml)

2.5 µl yeast RNA

5.5 µl 3 M NA-acetate (pH 5.6)

137.5 µl icecold 100 % EtOH

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

7. centrifuge 30 min at 14000 rpm at 4°C

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

-repetitive probes in 100 µl 60 % form/SSCP

-unique sequences in 50 µl 50 % form/SSCP

-cosmids, YACS in 50 µl TE

-libraries in 100 µl TE