Southern Transfer to Nylon membranes

Principle:

SoutherntransferistheclassicmethodusedtomoveDNA(usuallyintheformofrestrictionfragments)fromagarosegelstoasolidsupport,e.g.nylonmembrane.TheDNAisboundtothemembraneinthesameposition(relativetootherfragments)asinthegel.Theuseofamembraneinhybridizationexperimentsisfarmoreconvenientthantryingtohybridizeinagel.InaSoutherntransfertheDNAisfirstdenaturedinthegel,thenthegelisplacedonabuffer-saturatedspongeysurface,themembranepositionedontopofthegel,anddryblottingpapersareaddedontopofthemembrane.Thebuffermovesbycapillaryactionthroughthegelandintothepapersabove.ThedenaturedDNAinthegelmovespassivelywiththebufferandisstoppedbythemembrane.DNAispermanentlyboundtothemembranebybakingorcross-linkingwithUV.ThisprotocolisdesignedfortransferringDNAforRFLPanalysis.TheoptimumsizerangeofDNAfragmentsdetectedthiswayis2-25kb.

Timerequired:

3days

Procedure:

Day1

1. Foreachgeltoberun,melta250mlstockbottleof0.8%1XTAagaroseinthemicrowave(weighthebottlebeforeheatingandmakeuplostvolumewithdH2Oafteragaroseismelted).Coolto50degreesC;orprepare250mlof0.8%Tris-Acetate-agarosefollowingtherecipegivenhereunderSolutions.

2. Sealbothendsofagelcastingtraywiththelabelingtapeandplaceitonalevelsurface.Whentheagarosehascooledto50degreesC,pour250mlTAagaroseintothegelcastingtraywithoutformingairbubbles.Ifyouseeairbubbles,movethemtotheedgeorpopthemwithapipettipandplaceacombinthefirstsetofslotsonthegelcastingtray.Placea30-35toothcombinthefirstsetofslotsinthegelcast.Lettheagarosesolidifyatroomtemperatureforatleast30minutes.

3. Removethetape,thenplacethegeltrayintheelectrophoresisbox.Orientthewellstowardsthenegativeend(blackleads)andpour1XTAbufferintotheboxuntilthegelissubmerged.Pullthecombgentlyoutofthegel.(Ifgelsarepreparedseveralhoursinadvancedonotpullcombuntilyouarereadytoload.)

4. Loadthesamplesinthedesignatedlaneorders,changingpipetmentipswitheachsample.UselamBDaDNAcombinationMarkerheatedto50degreesCtomeltthe""stickyends"".(Formoreaboutthesemolecularweightstandardmarkers,refertoappendix)

5. Circulatethebufferusingalowspeedpump,e.g.aDIASpump,withthebufferflowingfromthenegativeend(blacklead)tothepositiveend(redlead)ofthegelbox.

6. Connecttheleadstothepowersupply,placethetimeronholdandadjustthevoltagetoaconstant40-45volts.Runthegelfor18-20hours(forScreeningblots)or20-24hours(forParent&Familyblots).

Day2

1. Tostainthegels,pour800-1000mlofdH2Ointoagelstainingbox,add15祃of10mg/mlethidiumbromide(EtBr),mixandimmersethegelinthestainingbox.Thestainsolutionshouldcoverthegelcompletely.Placeagelrestraineronthetraytoholdthegelinplace.

2. Shakegelsatroomtemperatureonaplatformshakerfor30minutes.Drainthestainsolution,rinsethegelindH2OtoremoveexcessEtBrandphotographthegelwithUVIlluminationusingtheFotodyneUV/MP-4camera.MarktheendsofthegelontheUVboxbymakingsmallcutstodefinewherethefinalcutsinthegelorSouthernblotwillbe,(e.g.toseparatetwofamiliesrunonthesamegel).Typicalexposuretimeisf5.6for1second.

3. TodenaturetheDNAinthegels,byshakegelsslowlyinafreshlypreparedsolutionof0.6MNaCl,0.2NNaOH(for1liter:200ml3MNaCl+20ml10NNaOHbroughtto1000mlwithdH2O)for30minutes.Checkfrequentlytobecertainthegelsstayimmersedinthedenaturant.

   Acommonproblemisthatifthegelsurfaceremainsdry,theDNAwillnotdenatureproperlyresultinginpoortransfer.Oftenthegelscanbekeptsubmergedwithasunkencleanglasstesttubepositionedontopofthegel.RinsegelsbrieflyindH2O.

4. Toneutralizethegels,shakeslowlyin1.5MNaCl,0.5MTris-HClpH8.0(for1liter:500mlof3MNaCl+500mlof1MTris-HCl).Checkfrequentlytobecertaingelsstayimmersed.
   

5. IfusingpreparedSouthernset-ups,removethetopblotblocksfromeachandreplacethemwithonesfreshlysoakedin10XSSC.Ifanewtransferset-upisneeded,wetblotblocksonebyonebysoakingthembrieflyin10XSSCandtransferringthemtoafreshblottingtray.Layerblotblockstoathicknessofabout11/2".Whenlayeringtheblotblocks,gentlyrollacleantesttubeacrossthetoptoremoveairbubblesbetweenblocks.

6. Fillthetrayswith10XSSCbufferuptothetopofthestack(butdonotimmersethetopofthestack).

7. Cut3MMWhatmanpapers(orusepre-cut3MMWhatmanpaper)tothesizeoftheblotblocks,individuallywetthemin10XSSC,andlayoneoneachstackofblotblocks.Removeairbubblesbyrollingthetesttubeasabove.

8. CutZetABInd(oranyothernylonmembraneusedasasolidsupport)slightlylargerthanthegels(e.g.8"x8")withapairofcleanscissors(weargloveswhenhandlingmembranesandprotectmembranesbyworkingonclean3MMpaper).Labelthemembranesacrossthetopwiththerespectivefamily/parentI.D.#,theenzymeandtheblotnumberusingadry-erasemarkerandcutacrossthelefthandtopcornerofthemembranesfororientationpurposes.

9. WetthenylonmembranesbrieflyindH2O,transfertoatraycontaining10XSSCandsoakfor20minutes.

10. Trimthegelsatthewells,thebottomandsides(ifnecessary)andslideontotheblottingtray.Removeanyairbubblesfromunderthegelswithglovedfingers.

11. Placethecorrectlylabeledmembraneontopofeachcorrespondinggelwiththenotchorientedtowardsthetoplefthandcorner.Removeairbubbles.Ifmorethanonefamilyisonagel,markthemembraneedgeswherecutswillbemadeafterthetransferiscomplete.

12. Foreachgel,wetanotherpieceof3MMWhatmanpaper(cuttothesizeofthegel)in10XSSCandplaceitonthemembrane.Removeairbubbles.CovertheexposedportionofthewetblotblockswithstripsofParafilmorSaranwrap(whichactasawickingbarrier).BecertainnottocoveranygellanescontainingDNA.Placeastackofdryblotblocks(~2cmhigh)ontopthe3MMWhatmanpaperandplaceasetofpapertowels(1-2incheshigh)ontop.

13. Placeagelrestrainerandaweightofabout500gontopofeachstackofpapertowels(a500mlbottleorflaskofwaterisapproximately500g).Refilltheblottingtrayswith10XSSCtowithin1/2inchfromthetopofthewetblotblocksandallowthetransfertogo6hourstoovernight.Checkthelevelof10XSSCoccasionally.

Day3

Takingdownthetransfers:

1. Discardthesoakedupperblotblocksandpapertowelsandtransferthemembranestoatraycontaining500ml2XSSC.Gentlyrubeachmembranewithaglovedhandtoremoveresidualagarose.SavethelowerblotblocksinthetrayandcoverthemwithSaranwrapforfutureuse.Labeltheblottingtraywiththedate.

2. Washtheblotsatroomtemperaturein2XSSCtwice,15minuteseachwash.

3. Airdrytheblotsbetweentwopiecesof3MMWhatmanpaperforatleast1hour.Baketheblots(betweenWhatmanpapers)at80癈inthevacuumovenfor1-2hours.

4. Washtheblotsinashakingwaterbathat65degreesCfor30minutesin500mlof0.1XSSC,0.5%SDS(2.5mlof20XSSC+25mlof10%SDSbroughtto500mlwithddH2O).Thisstepappearstoreducethenon-specificbackgroundoftenseeninthefirsthybridizationofblots.

5. Storetheblotswetasfollows:placeverywetinablotbagorseal-a-mealbagandsealthebagwithaT-barheatsealer.

6. Filloutanewblotrecordsheetforeachblot,tapethegelpictureonasheetofpaperwithalltherelevantgelconditionswrittendownandfilebothsheetsintheproperblotbookFiletheblotinitsrespectivefilefolder.

Solutions:

• 25XTris-Acetatebuffer:pH7.8:

  	for4liters	for20liters
  Trizmabase	484g	2420g
  EDTA,disodiumsalt	37g	185g
  Sodiumacetate.3H2O	136g	680g
  GlacialAceticacid	approx.166ml	approx.830ml
  addddH2Oto	4liters	20liters
  AdjustpHto7.8withGlacialAceticAcid,ifnecessary.

• 1XTAbuffer:

  In a 4L carboy, add 160ml of 25X TA and bring the volume to 4L with ddH2O.

• 3MNaCl:

  Pour 16 liters of ddH2O into a 20 L carboy. Slowly add 3 kg NaCl. Mix well. When the NaCl has dissolved, adjust the volume to 17.1 literswith ddH2O.

• 10NNaOH:

  Prepare in the chemical fume hood, and wear gloves and goggles:To a 4L carboy add 2L of ddH2O and slowly add 1600 g of NaOH pellets.
  Mixovernight.
  Adjustthevolumeto4LwithddH2O.Caution:Thisisanexothermicreaction.Thesolutiongetsveryhot!!
  1MTris-HCl,pH8.0:
  Pour 5 liters of ddH2O into a 20L carboy. Slowly add 2500 g of Trizmabase with a stir bar stirring vigorously and bring the volume to 18liters with ddH2O. Mix overnight.Add 1000 ml of concentrated HCl. Adjust the pH to 8.0 with more HCland bring the volume to 20.65 liters with dH2O.
  0.8%1XTAagarose:
  Weigh 2.0 g of agarose into each 500 ml bottle. Add 250 ml of 1X TA toeach. Weigh the bottles before heating and make up lost volume withdH2O after dissolving the agarose. Swirl the bottles to mix the agarosewell and either autoclave the agarose solution for 10 minutes or heatin a microwave oven on HIGH for 6-8 minutes. Keep bottle lids loosewhen heating the solution. Once the agarose is completely in solution,swirl each bottle gently to mix the agarose evenly. Place the bottlesin a 50 degrees C waterbath for 20-30 minutes before pouring thegel.
  10%SDS
  Dissolve 100g sodium dodecyl sulfate (SDS) in 500 ml of ddH2O, adjustvolume to 1000 ml and store at room temperature. Wear a face mask whileweighing out SDS.
  20XSSC(20liters)
  Dissolve 3504 g NaCl and 1760 g sodium citrate in 10 L ddH2O.(Use a 20 L carboy.) Adjust the volume to 20 L with ddH2O, and the pH to 7.4 with several drops of concentrated HCl.
  References:
  Southern,E.M.(1975)."DetectionofspecificsequencesamongDNAfragmentsseparatedbygelelectrophoresis."J.Mol.Biol.,98:503.
  Sambrook,J.,Fritsch,E.F.,andT.Maniatis.(1989).MolecularCloning-ALaboratoryManual(secondedition),ColdSpringHarborLaboratory,ColdSpringHarbor,NewYork.p.9.31-9.44.