78
MORIMASA MATSUTA AND MAYUMI MATSUTA
6. Remove the slides from the 100% ethanol.
7. Dry slides by touching the bottom edge of the slides to a blotter and wiping the underside of the slides dry with a paper
towel.
8. Air-dry.
Hybridization
9. Add the following to a 0.5 ml microcentrifuge tube at room
mix
temperature:
- 7).,ll of Master Mix
- 1).11 carrier DNA (herring testis DNA or Cot-l DNA:500).1g/
ml)
- 2).11 probe (biotinized or digoxigenin-labeled DNA probe:
diluted if necessary)total 10).11 , for each target area
Note: Master Mix (of Hybridization) volume is 70% ofthat used
in the final hybridization mix. The remaining 30% of the volume
are filled with probe(s), carrier DNA, and water if needed. Used
in this way Master Mix gives a hybridization mix that is 55%
formamide/lO% dextran sulfate/lXSSC. Hybridization buffer,
which contains both Master Mix and carrier DNA, is commercially available from Vysis or Oncor.
Typical probe concentrations are 2 ).1g/ml for genomic DNA and
OA).1g/ml for chromosome-specific repetitive sequences. A typical hybridization mix for a 18-20mm square area on a slide might
be 10 ).11.
10. Centrifuge tube for a few sees.
11. Vortex and re-centrifuge.
12. Place tube in a 74°C water bath for 5 mins.
13. Remove tube from water bath.
14. Place tube on ice until ready to apply hybridization mix to
target DNA on slides.
Note: If slides are ready, you can apply hybridization mix immediately to target area.
Hybridization 15. Apply 10 ).11 of probe mix to slide. Apply 18-22 mm square
glass coverslip on placing hybridization mix.
16. Seal coverslips with rubber cement.
MORIMASA MATSUTA AND MAYUMI MATSUTA
6. Remove the slides from the 100% ethanol.
7. Dry slides by touching the bottom edge of the slides to a blotter and wiping the underside of the slides dry with a paper
towel.
8. Air-dry.
Hybridization
9. Add the following to a 0.5 ml microcentrifuge tube at room
mix
temperature:
- 7).,ll of Master Mix
- 1).11 carrier DNA (herring testis DNA or Cot-l DNA:500).1g/
ml)
- 2).11 probe (biotinized or digoxigenin-labeled DNA probe:
diluted if necessary)total 10).11 , for each target area
Note: Master Mix (of Hybridization) volume is 70% ofthat used
in the final hybridization mix. The remaining 30% of the volume
are filled with probe(s), carrier DNA, and water if needed. Used
in this way Master Mix gives a hybridization mix that is 55%
formamide/lO% dextran sulfate/lXSSC. Hybridization buffer,
which contains both Master Mix and carrier DNA, is commercially available from Vysis or Oncor.
Typical probe concentrations are 2 ).1g/ml for genomic DNA and
OA).1g/ml for chromosome-specific repetitive sequences. A typical hybridization mix for a 18-20mm square area on a slide might
be 10 ).11.
10. Centrifuge tube for a few sees.
11. Vortex and re-centrifuge.
12. Place tube in a 74°C water bath for 5 mins.
13. Remove tube from water bath.
14. Place tube on ice until ready to apply hybridization mix to
target DNA on slides.
Note: If slides are ready, you can apply hybridization mix immediately to target area.
Hybridization 15. Apply 10 ).11 of probe mix to slide. Apply 18-22 mm square
glass coverslip on placing hybridization mix.
16. Seal coverslips with rubber cement.