Northern Hybridisation
1. Unwrap the filter and place on a membrane support (RNA side facing
you).
2. Roll the filter away from you and place into a hybridisation bottle.
3. Add 15mL of pre-warmed Ultrahyb.
4. Place the bottle in hybridisation oven at 42oC for a minimum of 30
minutes.
5. After 30 minutes add your radioactive probe to the filter and hybridise
for 18 hours at 42oC.
WEAR SAFETY GOGGLES
6. After 18 hours, carefully discard the solution into the radioactive sink
with tap running.
7. Add 30mL 2xSSPE/0.1% SDS/0.1% pyrophosphate and place the
bottle in hybridisation oven for 30 minutes.
8. Carefully discard the solution down the radioactive sink.
9. Add 30mL 0.2xSSPE/0.1% SDS/0.1% pyrophosphate and place the
bottle in hybridisation oven for 30 minutes.
10. Carefully discard the solution down radioactive sink.
11. Behind a screen remove filter into a tray.
12. Place the filter onto a piece of towel and allow to air-dry.
13. Check with the counter and if too much background return to
hybridisation bottle.
14. Add 30mL 0.1xSSPE/0.1% SDS/0.1% pyrophosphate and incubate
for 10 minutes. (Repeat wash if necessary)
15. Repeat steps 10 to 12.
16. Wrap the filter in Saran wrap and place in a cassette.
17. In the darkroom under safelights place an X-ray film against the filter.
18. Place cassette on designated shelf in -80oC freezer.
19. Place the tray, membrane support and bottle in the radioactive sink
and flush with running water.
20. After 30 minutes check the bottle etc. for radioactivity, if clear place
on draining to dry.
21. If anything is still radioactive then place in a tray of 2% decon and
leave soaking overnight in the sink.
22. 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.