NICK TRANSLATION OF dsDNA WITH BIOTINYLATED OR DIGOXIGENIN dUTP

互联网2013-09-06

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<center> <font>NICK TRANSLATION OF dsDNA WITH BIOTINYLATED OR DIGOXIGENIN dUTP</font></center>

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