Southern Hybridazation

Hybridization

Materials

PrehybridizationSolution	(10-50mL).Mixbyrotatingat37°C:
	Startwith50mLStark"sSolution(with50%formamide)
	+0.5%non-fatdrymilk(0.25gm)
	+1%SDS(0.5gm)
	+8%dextransulfate(4gm,SigmaD-6001)
42°Cshakingwaterbath
100°CHeaterblock
32P-labledprobe	use10-20ngDNA/filter,orabout10millionCPM/filterforSouthern"s
Nylonfilter	DNApreviouslyblottedontofilter(e.g.GeneScreen+)
HybridizationChamber	useGlassbelljarfor9cmdiskfilters
	orSealAMealbagandglasstrayforlargerfilters

Procedure

1)MixPrehybridizationSolutionuntildissolved.Incubatefiltersat42°CinPrehybsolutionineithertheglassbelljaroraSealAMealbagfor1-4hrs.

2)Turnonheaterblockto100°C.

3)Transferprobeto1.5mLEppendorftubewithapinholeinthecapandaddH2Otobringthetotalvolumeto150µL.DenaturetheDNAbyheatingto100°Cfor5min.

4)Transferprobetothefilterandchamber.MixwellwiththePrehybsolution.Resealthebagorputalidandweightonthebelljar.Incubatewithshakingat42°Covernight.

WashingFilters

Materials

20xSSC(3MNaCl,0.3MCitrate,pH7.4)

65°Cshakingwaterbath

Whatman3Mpaper

Saranwrap,florescentstickers

AutorADIographcasetteandintensifyingscreens

X-rayfilm(KodakBioMax8x10",Cat829-4985)

1)DiscardtheradioactiveHybridizationsolutionintoabsorbedliquidwastecontainer.Rinsethefilterwith1xSSC,0.1%SDSanddiscard.

2)Washthefilterin1xSSC,0.1%SDSinashakingwaterbathfor1hour.Meanwhileallowthewaterbathtoheatto65°C.Changewashsolution2xduringthehour.

3)Washthefilterin0.1xSSC,0.1%SDSat65°Cfor10to20minutes.MeanwhilemonitortheCPMonthefilterwithaGeigercounter.Stopwashingtheblotwhenthecountsarereducedto2-4xbackground.

4)BlotthefiltersonWhatman3MMpaper.WrapthefilterinSaranwrapandplaceglowinthedarkstickersfororientation.InadarkroomplacethefilterinanautoradiographcasettethenapieceofX-rayfilm,andthenanintensifyingscreen.Ifaweaksignalisanticipateddothisincompletedarkness(withthesafelightsoff).

5)Placethefilmcasetteat-70°C.ForclonedDNAandhighS.A.probesexposefor2-4hours.FormammaliangenomicDNASouthernblotsexposethefilmfor18-72hrs.

6)Removethefilmcasetteandallowittowarmfor30min.Removethefilminthedarkroom(forweaksignalsdothiswiththesafelightsoff)anddevelopinaprocessororindevelopingtanksasfollows:X-raydeveloper:2min.Waterwash10sec.Fixer:2min.Waterwash5min.Airdrythefilm.