Prehybridization and hybridization procedures

Use RNase precautions and RNase-free solutions prepared using diethyl pyrocarbonate (DEP)-treated dH20.

Protocol 6.

Method

11. Float the dry membrane, RNA side up, on DEP-treated dH20. When completely wetted, submerge the filter.

12. Place into a heat-sealable plastic bag.

13. Pipette 10 ml of pre-hybridization/hybridization solution into the bag.

14. Heat-seal the bag, then cut a corner off, and careful expel all bubbles. Seal.

15. Pre-hybrldize submerged in a 42°C water bath with gentle shaking for 6 h up to overnight. Occasionally, massage the bag to ensure uniform distribution of solution.

16. Cut a corner of the bag and expel the pre-hybridization solution.

17. Pipette into the bag 10 ml of pre-warmed (42°C) hybridization solution containing 106 cpm per ml of 32P-labelled DNA probe.

18. Carefully expel bubbles, catching any radioactive fluid on paper towels. Heat-seal the bag and test for leaks.

19. Incubate in the 42°C bath overnight with gentle shaking. Occasionally massage the bag to mix the solution.

10. Expel radioactive hybridization solution into an appropriate container. Remove the blot and wash for 20 min at room temperature in 2 x SSC/0.1% SDS (200-250 ml per blot) with shaking.

11. Wash at 68°C in pre-warmed 2 X SSC/0.1 % SDS for 15 min with vigorous agitation.

12. Rinse briefly (3-5 minutes) in 2 X SSC (room temperature) to remove excess SDS.

13. Dry the membrane briefly, RNA side up, on 3M paper but leave slightly damp.

14. Cover in plastic wrap and expose to X-ray film using intensifying screens at -80°C.

15. If the background is high, re-wet the membrane in 2 X SSC and wash at 68°C for 15-30 min in 1 x SSC/0.1% SDS. Expose to film. If the background is still high, try successive washes (with exposures to film in between) using 0.5 x and then 0.1 x SSC with 0.1 % SDS.

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