FISH Protocol
- Labeling probe with nick translation
Recipe:
1 µg DNA
5 µl 10x nick translation buffer
5 µl dNTP (0.5 mM dATP, dCTP, dGTP, 0.1 mM dTTP)
5 µl DTT (100 mM)
2 µl Biotin-dUTP (1 nmol/µl)
9 µl Dnase I (1 µg/µl)
1 µl DNA polymerase I (10 U/µl)
ddH2O to 50 µl
Incubate at 15°C for 2 hours.
Add 5 µl 0.5 M EDTA to stop the reaction.
Run 4.5 µl + 1 µl BFB on a 2% agarose gel, the fragment size should be 200-500 bp.
- In situ Hybridization
EtOH precipitate labeling probe:
2.5 µl nick-translation probe
2 µl Human Cot-1 DNA
8 µl SS-DNA
1 µl 3M NaAc
30 µl 95% EtOH
sit on dry-ice for 15 min.
Centrifuge at 14000 rpm for 15 min at 4°C.
Discard the supernatant
Wash the pellet with 30 µl 70% EtOH at 14000 rpm for 5 min.
Discard the supernatant, dry in vacuum for 5 min.
Dissolve in 11 µl 50% hybridization mixture, vortex, spin down.
Shake 15 min. Denature 5 min at 75°C. Prehybridization 40 min at 37°C.
**Slide treatment
Denature slides with 50 µl 70% denaturing solution for 5 min at 75°C.
Dehydrate in –20°C EtOH-series (70%, 85%, 95%) 5 min/each.
Let the slides dry in air.
**Hybridization
Preheat the slides at 42°C.
Add 1 µl chromosomespec-centromeric probe to the prehybr-probe.
Place the mixed probe solution on the slides.
Cover with a coverslip and seal with rubberglue.
Place the slide in a dark moister chamber.
Incubate over night at 37°C.
**Washing and detection
Wash the slide in 2 x SSC for 5 min at 72°C.
Amplify with biotin-antibody and detect with FITC-avidine 3 times.
Dehydrate in 70%, 85%, 95% EtOH.
Let air dry in darkness.
Stain with DAPI and the slide is ready for the microscope.
