PreparationofNestedDeletions

1)Needtocut10µgofplasmidintwospots("A"and"B")inpolylinker."A"cutis"itharestrictionenzymewhichgivesa3"recessedend(exonucleasesensitive)nexttoinsert.Since3"recessedendcanberelativetocodingornoncodingstrand,"A"cutcanbeoneithersideofinsert.[Forsequencingbothstrands,willneedtomakenesteddeletionsfrombothsides.]Aftera1hr(100µl)"A"restrictiondigest37°C,takea2.5µlaliquotforgelanalysis,thenadd10µlof3Msodiumacetate,pH5.2,and200µlof100%ethanol.Spinfor15minat4°C.Removesupernatant.Add500µlof70%ethanol,vortexgently,thenspinfor5minat4°C.Removesupernatant,addmore70%ethanol,vortex,spin,pullofethanol,thenletDNApelletairdryfor5min.

2)MakepelletupinddH2Oanddo"B"restrictiondigestfor1hr(37°C)in100µl."B"cutiswitharestrictionenzymewhichgivesa3"protrudingend(threebaseorgreater;exonucleaseresistant).Save2.5µlofdigestforgelanalysis.

3)Add1.35µlof1/3diluted1MMgCl2togiveafinalconcentrationof10mMMg.Heatfor10minat70°C

4)Totiterphagelibrary,thawphage,andaliquotifitisanewlyreceivedlibrary.Add3µlof10folddilutionsofphage(undil.to1/2000;dilutein10mMMgSO4;bestdilutionfor#1055inExp.1727was1/1000-1/1333)to600µlofdilutedcells(fromstep3))insterilebluetop(#14-956-4A)tubes.Mixbyshakingorgentlyvortexing,thenincubatefor15minat37°Conashaker.Duringthistime,melttopagaroseandhavecoolingto47°C(checktemperatureonoutsideofbottlewithBioRadtemp.indicatorstrip).

5)Add6.5mlof47°Ctopagarose(keeptopagaroseina50°Cwaterbathonthebenchbytheflametokeepfromsolidifying)tothefirsttube,mixbyinversionseveraltimes,thenquicklypourontothecenterofa150mmNZYplate.Quicklyswirltopromoteevendistribution.Repeatwiththenexttubes.Afterallplateshavebeenpouredandhardened,invertandincubateat37°C.Count#ofplaquesafter12-16hrs.

6)Usingsamehostcellsfrom3),dilutephagetogive15,000-20,000cfu/150mmplate.Do20plates,asper4)and5).Incubate12-16hrsat37°C.

7)Putplatesat4°Cfor2hrs.Duringthistime,preparesolutionstobeusedinsteps7)-9),includingprehybridizationandhybridizationsolutionswhichshouldbeplacedina42°Cincubatortowarm.Labelfirst137mmfilter(Stratagene#420107)withfinetipindelIBLeMarkertocorrespondtoplate.Holdingfilteratedgewithbluntendedforceps,placefilterontoplaquesurface.Markfilterin3asymmetriclocationsbystabbingthroughitandagarwith18gaugeneedleattachedtoasyringecontainingwaterproofblackink.After1min,pullofffilterandimmerseDNAsideupfor1-5mininaglasstraycontaining500mlof0.5NNaOH,1.5MNaCl(25mlof10NNaOHand150mlof5MNaClmadeupto500mlinddH2O).After10filters,willneedreplacethiswithfreshdenaturingsolution.

8)Removeandtransfertoneutralizingsolutionfor5minconsistingof500mlof1.5MNaCl,0.5MTris,pH7.4(150mlof5MNaCland250mlof1MTris,pH7.4madeupto500mlinddH2O).

9)Rinsefilterintwosuccessive2xSSCbaths(400mleach;80mlof20xSSCmadeupto800mlinddH2O),thenplaceDNAsideuponfresh3MMWhatmanorbenchtoppapertodry(1hr).Whenallfiltersaredry,sandwicheachbetween3MMWhatmanpaper,andbakeat80°Cfor2hrsundervacuum(needdryiceintrap).

10)Allowfilterstocool,thenindividuallywetin2xSSC(ina150mmpetridish;needabout50ml),followedbyimmersioninprewarmedprehybridizationsolutioncontainedalsoina150mmpetridishsupportedinaplasticbox.Puttoponbox,andputinincubatorat42°Cfor2hrswithgentleswirling.Afterprehyb,individuallytransferfiltersintoa150mmpetridish(inplasticbox)containinghybridizationsolution.Placetoponboxandputinincubatorat42°Covernightwithgentleswirling.

PrehybridizationSol"nHybridizationSol"nddH2O15.3mlddH2O15.3mlFormamide15mlFormamide15ml20xSSPE12.5ml20xSSPE12.5ml20%SDS0.25ml20%SDS0.25ml50xDenhardt"s5ml50xDenhardt"s5mlSalmonSp.DNA2mlSalmonSp.DNA2mlPolyA5µlPolyA5µl32CDNAprobe50-100µl

(note:SalmonSpermDNAstockis5mg/ml;PolyAstockis10mg/ml;makeupprehybandhybsolÕnsin50mlconicals)11)Removefiltersindividuallyinto400mlofroomtemperature2xSSC,0.1%SDS(40mlof20xSSCand4mlof20%SDSmadeupto800mlinddH2O).Washfor5minonrotator.Dotwoadditionalwashes.Transferfiltersto400mlofprewarmed(42-65°C;use42°Cforwheat-mouseand65°Cformouse-mousehybridizations)0.1xSSC,0.1%SDS(4mlof20xSSCand4mlof20%SDSmadeupto800mlinddH2O).Washfor20minonrotator.Doatotalofthree20minwashes.

12)Allowfilterstodryonfreshbenchpaper,thenplaceonWhatmann3MMpaperinalargex-raycassetteholder.Coverwithplasticwrap.Underredlightinthedarkroom,placex-rayfilmon,followedbyanenhancingscreen.Closeupandexposeovernightat-70°C.Thenextdaydevelopfilm.Copylocationofpositiveplaquesandthreeasymmetricinkmarksontoaclearacetatesheet.Placeacetatesheetonbottomoforiginal150mmplateandidentifypositiveplaques.

13)Pickpositiveplaquesbystabbingwithaplastictransferpipet,andsquirtinto1mlofSMcontainingadropofchloroform.Letsitfor1-2hrsatRTorovernightat4°C.Vortexgently.Dilute1/100(2µlupto200µl)in10mMMgSO4.Add50µlofdilutedsamplewith600µlofXLI-BlueMRF"hostcells(OD600=0.5),andmix,thenincubatefor15minat37°Conashaker.Add6.5mlof47°Ctopagarose,mixbyinversionseveraltimes,thenquicklypourontothecenterofa150mmNZYplate.Quicklyswirltopromoteevendistribution.Incubateat37°C.Repeatsteps7)-12).Thisisthe2°screen.14)Forthetertiaryscreen,pickpositiveplaques,asin13).Dilute1/1500sothatplaqueswillbewellseparated.Mixwithhostcellsasaboveandplatewithtopagarose.Repeatsteps7)-12).Allplaquesshouldnowbepositive.Pickawell-separatedplaquefromeachofthetertiaryscreenclones.Placein500µlofSMcontaining20µlofchloroform,vortexgentlyandincubatefor2hratRT,orstoreat4°C.GotoÔExcisionofClonesaspBluescriptfromlZAPII".NOTE:todoexcisionexperimentonsamedayastertiaryplaquepicking,needtohavepreparedovernightculturesofXLIBlueMRF"andSOLR(establishedfromfreshstreakedplates).