Standardhybridizationtechnique

Youwillneed:

6xSSC(0.9MNaCl,90mMsodiumcitrate,pH7)10%SDS50mg/mlheparinsulphate(Sigma)

Standardhybridisationsolution:6xSSC,500ug/mlheparin,1%SDS.

N.B:AmodificationusedforthehybridisationbufferwhichcanalsogiveextremelygoodresultsisavariationonthatdescribedbyChurchandGilbert,1984.Thebuffercontains7%SDSand0.25MNa₂HPO₄,pH7.2.

Pre-hybridisationandhybridisationofblotsiscarriedoutusingaHybaidrotaryhybridisationovenandbottlesat60°C(NorthernblotsorRNAdotblots)or65°C(plaquehybridisationsorDNAdotblots).

1)Placemembraneinthehybridisationbottlewiththeminimumamountofhybridisationsolution(0.1ml/cm2membrane).

2)Pre-hybridiseattheappropriatetemperatureforatleast3hours.

3)BoiltherADIolabelledprobefor3minutestodenaturetheDNA.

N.B:Careshouldbetakenwhenhandlingradioactivesources.Appropriateprotectiveclothingandradiationmonitoringequipmentshouldbeusedatalltimes.

4)Addprobetothehybridisationbottletogiveafinalconcentrationofapproximately1-5x10⁵cpm/ml(byCerenkovcounting)ofhybridisationfluid.Allowhybridisationtocontinuefor6hoursormore,typicallyovernight.

5)Oncehybridisationiscomplete,thehybridisationsolutioniscarefullyremovedandthemembranewashed,sequentially,inthebottleinthefollowingfashion:-

3x10minutesin50ml2xSSC,0.1%SDSat65°C3x20minutesin50ml0.2xSSC,0.1%SDSat65°C2x10minutesin50ml0.1xSSCat37°C

N.B:Optimumtemperaturesforeachprobe-targethybridisationreactionwillobviouslyhavetobedeterminedempirically.

6)Afterwashing,removethemembraneandairdry.WrapthemembraneinSaranWrapandautoradiographat-80°C.

Ifthemembraneisrequiredforre-probing,theairdryingstepshouldbeomittedandthemembraneautoradiographedwet.

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