AnalysisofGenomicDNAbySouthernHybridization

Probepreparation

1. SelectseveralindependentBACclonescontainingthesameinsertsthatwillbeusedasaprobe.
2. ConfirmtheBACcloneintegrityusingbycomparingHindIIIdigestionpatterns.
3. Purificationofthewholeinsertsfromthoseclones.(NotIdigestion,PFGErunning)
4. Heatgelfragment(FMCSeaPlaqueGTGAgarose)containingtheinsertat65°Cfor10min.Transfer25µltoanew1.5mltube,boilfor5min.andchillonice.
5. Addthelabellingpre-mixtothetube(AmershamMultiprimeDNAlabellingsystemRPN16000Z).Keepovernightatroomtemperature.

    pre-mix: 4.0µl dCTP 4.0µl dGTP 4.0µl dTTP 5.0µl 10x buffer 5.0µl primer/BSA 2.5µl [32P]dATP2.0µlKlenow

6. RemoveunincorporateddNTPbyspincolumn(PharmaciaSephadexG-25)

Filterpreparation

1. DigestanappropiateamountofBACDNAandhighmolecualrweightgenomicDNA.ForSouthernhybridization,approximately10µgofDNAshouldbeloadedintoeachslotofthegelwhenprobesofstandardlengthandhighspecificactivityareusedtodetectsinglecopysequences.

    Example: Human genomic DNA (Clonetech) 1) Mix DNA 100µl(10µg), NEBuffer240µl,HinIII(20u/µl)2.5µl,incubateat37°Cfor2-2.5hours2)Add800µlabsoluteEtOHtothetube,andinvertforseveraltimes.3)Vortexwithhighestspeedfor2min.4)Spinatthehighestspeedfornotlongerthan5min.5)RemovetheEtOHcompletelyanddissolvewith25µl2xBPB.

2. LoadboththeBACcloneandhumangenomicDNAonto0.7%gelin0.5xTBE.ThegelshouldberunveryslowlytopreventsmearingoftheDNAfragments.
3. Afterelectrophoresis,stainthegelwithEtBrandphotographir.PlaceatransparentruleralongsidethegelsothatthedistanceofmigrationoftheDNAbandscanbereaddirectlyfromthephotograph.
4. Capillarytransferthegeltothepositivelychargednylonfilter(Schleicher&Schull,NYTRANPLUS,0.45µmporesize).

    Denaturation and transfer solution: 0.5M NaOH + 1.5M NaCl Neutralizing solution: 0.5M Tris + 1.5M NaCl (pH7.4)

5. TofixtheDNAonthefilter,bakethemembranefor1.5-2hoursat80°Cinthevacuumoven.

Filtersupression(pre-hybridization)

1. PlacethefilterintheFalcon#2098plasticcentrifugetube.
2. Add4mlhybridizationsolutiontothetube(1MNaCl.0.05MTris·ClpH8.0,0.5mMEDTA,1%SDS,and10%Dextran).
3. Boil40µl(10mg/ml)coldnonspecificDNA(Sigma,humanplacentalDNA)in400µlTE(pH8.0)for5min.andaddtothehybridizationbuffer.
4. HybridizethefilterwithcoldnonspecificDNAat65µCforovernightinahybridizationincubator(RobbinsScientific).

Probesupression

1. Preparethesupressionmixture

    10µl labelled BAC probe 40µl cold nonspecific DNA 50µl poly dG-dT/dA-dC (0.25mg/ml) (Pharmacia) 200µl 1.5x SET (1x SET: 0.6M NaCl, 0,02M EDTA, 0.2M Tris·Cl [pH8.0], 2% SDS, 0.1% sodium pyrophosphate)

2. Boilfor5min.
3. Keepat65°Cforatleast3.5hours.
4. Addthismixturetothepre-hybridizedfilter,keepat65°Covernight.

Washing

Washthefilterseveraltimesat65°Cwithsufficientvolumesof0.1xSSC/0.1%SDS(20min.eachtimeforatleasttwice).

Exposure

ExposethefilterwithphosphoimgaerorX-rayfilm(KodakBIOMAXMSfilmandintensifyingscreen)untilsignalisvisIBLe.