Southern Hybridisation
1. Unwrap the filter and place on a membrane support into a shallow tray
containing a small amount of pre-warmed hybridisation solution.
2. Roll the filter away from you and place into a hybridisation bottle.
3. Add 30mL pre-hybridisation solution.
4. Add 500uL denatured Herring sperm DNA (boil for 5 minutes, incubate on
ice for 2 minutes).
5. Place bottle in hybridisation oven at 65oC for a minimum of 4 hours.
6. After 4 hours add your radioactive probe to the filter and hybridise for 18
hours at 65oC.
WEAR SAFETY GOGGLES
7. After 18 hours, carefully discard the solution into the radioactive sink with
the tap running.
8. Add 30ml 2xSSPE/0.1% SDS/0.1% pyrophosphate and place the bottle in
hybridisation oven for 30 minutes.
9. Carefully discard the solution down the radioactive sink.
10. Add 30mL of 0.2xSSPE/0.1% SDS/0.1% pyrophosphate and place the
bottle in hybridisation oven for 30 minutes.
11. Carefully discard solution down radioactive sink.
12. Behind a screen remove the filter into a tray.
13. Place the filter onto a piece of towel and allow to air-dry for about 30
minutes.
14. After 30 minutes check with counter and if too much background return to
hybridisation bottle.
15. Add 30mL 0.1xSSPE/0.1% SDS/0.1% pyrophosphate and incubate for 10
minutes.
16. Repeat steps 11 to13.
17. Wrap the filter in Saran wrap and place into a cassette.
18. In the darkroom under safelights place X-ray film against filter.
19. Place cassette on designated shelf in -80oC freezer.
20. Place the tray, membrane support and bottle in the radioactive sink and
flush with running water.
21. After 30 minutes check the bottle etc. for radioactivity, if clear place on
draining board to dry.
22. If anything is still radioactive then place in a tray of 2% decon and leave
soaking overnight in the sink.
23. The next day remove the radioactive contents from the tray, rinse with
water and check to see if clear, if clear place on draining board to dry, if still
radioactive return to tray containing decon and soak again.