6 In Situ Hybridization for DNA: Fluorescent Probe
75
7. Air-dry.
8. Add the following to a 0.5 ml micro centrifuge tube at room Hybridization
temperature:
mix
- 7J..tl of Master Mix
- 1111 carrier DNA (herring testis DNA or Cot-l DNA:500llgf
ml)
- 2111 probe (fluorochrome-directly-Iabeled DNA probe: diluted if necessary)total lOlli, for each target area.
Note: Master Mix (of Hybridization) volume is 70% of that 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% formamideflO% dextran sulfateflXSSC. To simultaneously hybridize up to three probes, each labeled with different fluorochromes, add 1 III of each probe. Add purified water to bring
the combined volume of probe and water to 3 Ill.
Hybridization buffer, which contains both Master Mixture and
carrier DNA, is commercially available from Vysis or Oncor.
Typical probe concentrations are 2 Ilgfml for genomic DNA and
O.4llgfml for chromosome-specific repetitive sequences. Use 2 - 3
III of hybridization mix fcm 3 • Thus a typical hybridization mix
for a 18-20mm square area on a slide might be 10 Ill.
9. Centrifuge tube for a few sees.
lO. Vortex and re-centrifuge.
11. Place tube in a 74°C water bath for 5 mins.
12. Remove tube from water bath.
13. 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.
14. Apply 10 III of hybridization mix to slide. Apply 18 - 22 mm Hybridization
square glass coverslip on placing hybridization mix.
15. Seal coverslips with rubber cement.
16. Place slides in a prewarmed humidified box and place box in
a 37°C incubator for 6-16 hours.
75
7. Air-dry.
8. Add the following to a 0.5 ml micro centrifuge tube at room Hybridization
temperature:
mix
- 7J..tl of Master Mix
- 1111 carrier DNA (herring testis DNA or Cot-l DNA:500llgf
ml)
- 2111 probe (fluorochrome-directly-Iabeled DNA probe: diluted if necessary)total lOlli, for each target area.
Note: Master Mix (of Hybridization) volume is 70% of that 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% formamideflO% dextran sulfateflXSSC. To simultaneously hybridize up to three probes, each labeled with different fluorochromes, add 1 III of each probe. Add purified water to bring
the combined volume of probe and water to 3 Ill.
Hybridization buffer, which contains both Master Mixture and
carrier DNA, is commercially available from Vysis or Oncor.
Typical probe concentrations are 2 Ilgfml for genomic DNA and
O.4llgfml for chromosome-specific repetitive sequences. Use 2 - 3
III of hybridization mix fcm 3 • Thus a typical hybridization mix
for a 18-20mm square area on a slide might be 10 Ill.
9. Centrifuge tube for a few sees.
lO. Vortex and re-centrifuge.
11. Place tube in a 74°C water bath for 5 mins.
12. Remove tube from water bath.
13. 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.
14. Apply 10 III of hybridization mix to slide. Apply 18 - 22 mm Hybridization
square glass coverslip on placing hybridization mix.
15. Seal coverslips with rubber cement.
16. Place slides in a prewarmed humidified box and place box in
a 37°C incubator for 6-16 hours.