Pre-hyb/hybforRADIoactiveProbes
Stuffyouneed:50XDenhardt"sreagent:1%Ficoll1%Polyvinylpyrrolidone1%BovineSerumAlbuminPrehyb/hybsolution:5XSSC50%formamide100µg/mlSSDNA(heatdenaturedat95degC)5XDenhardt"s0.2%SDSBlotWash1:2XSSC0.1%SDSBlotWash2:0.1XSSC0.1%SDSStrippingSolution5mMTris-HCl,pH8.02mMEDTA,pH8.00.1XDenhardt"s1.Aftertransfer,crosslinkandhybridizefor1hourinhybridizationbufferat42degC.2.Boilprobein500µLhybridizationbuffer.Coolonice.3.Addprobetoblotandincubateovernightat42degC.4.Removeblotfrombottleanddothefollowingwashesinapyrexdish:5minRTBlotWash115min55degCBlotWash2Checkradioactivityassociatedwithcornerofblot.Ifstillhot:15min55degCBlotWash2.5.Wrapblotinplasticwrapandputdowntofilm.Cassetteat-70degC.6.Tostrip,addstrippingsolutionandincubateat65degCfor2hours,oruntilradioactivityisremoved.7.StoreblotbetweentwosheetsofWhatmanat4degC.