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.Standardhybridizationtechnique
Thestandardsolutiontypicallyusedforbothpre-hybridisationandhybridisationisbasedonthatgiveninManiatisetal.,(1982)withbothDenhart"ssolutionandheterologousDNAbeingreplacedbyheparin(SinghandJones,1984).
