DeterminetheOD600andcorrelatethecelldensityfromthechart.Setupfour100mLYPDculturesatthefollowingdensities:0.7x105,1x105,and3.0x105cells/mLGROWTHOFCELLSGrow100mLofcellstoOD600=0.7-0.8at23oC.Add5-10ulofBME,15minutesbeforespin.Harvestcellsintwo50mLconicaltubes,spin1.5-2.0Kfor8-10min.SPHEROPLASTING.ResUSPendcellsin4mLYWB.*NOTE:Ifcellshavebeenarrestedwithnocodazoleoralphafactor,includetheinhibitorintheYWB.Letsitatroomtemperaturefor2-3min.Addoxalyticase[12uL1mg/mlstock].Incubateat23oCwithgentleshakingtoformspheroplasts.Spheroplastingshouldbecompletewithinonehour;avoidincubationslongerthan1h,15minTransferspheroplastedcellstoaTOMYtube.Spinat3.5Kfor7min.Resuspendcellsin4mLYWB(containing5%glyceroland1mMPMSF[0.2Mstockin100%EtOH])bygentlypipettingupanddown,usinga1mLpipet.Spinat3.5Kfor7min.Gentlyresuspendcellsin2.5mL1xEBB(containing5%glyceroland0.4mMPMSF).Allowcellsto"swell"for10min.atroomtemperature,thentransfertohomogenizer.[RinsedouncewithH2O,EtOHtoclean.RinsedouncewithEBBbeforeusing.]Homogenizecellswith5pestlestrokes(upanddownisonestroke).Avoidintroducingair.TransfertocleanTOMYtube.Add150uL5MNaCl(finalconcentration~0.3M).Incubateatroomtemperaturefor5min.Add5mL1xEBB-plus(containing5%glycerol,0.1mMDTTand150ug/mLBSA).Thus,finalconcentrationsin7.5mLlysateare0.1MNaCl.Incubateatroomtemperaturefor45min.Meanwhile,thawmicrotubule"seeds"andputat37oCfor30min.Addtaxolto10uM(e.g.1ul0.13mMtaxolto15ulMTseeds)andincubateanadditional15min.Remove250ulsampleforTOTALMATERIAL("1T").Keepthistubeatroomtemperature,soastobecomparabletotheothersamples.DivideremaininglysatebetweenEppendorfstubes.CLARIFYLYSATESSpineppendorftubesat15Kfor20min.Poolsupernatantsina15mLconicaltubeandmixgently.Aliquotlysatetomicrofugetubes(between800uland1ml).Spinmicrofugetubesat15Kfor20min.Remove250ulTOTALSUPERNATANT("1TS")toafreshtube.Remove500ulclarifiedlysatefromeachmicrofugetubetoafreshtube.Add5ul1mMtaxol(inDMSO)toeachtube-finalconcentrationis10uMtaxol.Mixgently.Forastandardtitrationofbindingactivity,addmicrotubulestoeachtubeindecreasingamount.Normally,8,4,2,1,0.5and0uls(MTsmadefrom~3-6mg/mlPCbovinetubulin).For0.5ul,makea10-folddilutionofMTsinBRB80/30%glycerolbuffer(containing10uMtaxol).Allowbindingtoproceedatroomtemperaturefor15min.Pelletmicrotubule/minichromosomemixat15Kfor8min.ItshouldbepossIBLetoseethelargerMTpellets.Remove250ulofeachsupernatanttofreshtube.ThesearetheSUPERNATANTFRACTIONS.AspirateallresidualliquidwithdrawnoutPasteurpipets.ResuspendMTpelletsin250ulof1xEBB(containing0.1mMDTT,100mMNaCl,5%glyceroland1mg/mlBSA,butNOPMSF!).ThesearethePELLETFRACTIONS.NOTE:Ultimately,PELLETswillbe2xconcentratedrelativetoSUPs.Add250ulof2xASSAULTbuffer(containingtRNAandøX174)toallsamples.Removeproteinbyadding20ulsofProteinaseKsolution(15mg/mlfromBoehringerMannheim)toeachtube.Incubatetubesat50oCfor~1-1.5h(longerisbetter).AfterProteinaseKtreament,precipitateDNAbyadding250ul6MNH4OAcand700mlisopropanol.Storeat-20oCovernight.SpindownDNAat15K,30min.4oC.Carefullyaspirateordecantsupernatants,removingasmuchsupernatantaspossible.NOTE:The"PELLETSAMPLE"precipitatedpelletsareoftensmall.WashallDNApelletswith100-200ul70%ethanol.Spinbriefly(~5min)andaspiratetheethanolsupernatant.AllowDNApelletstoairdry.Donotinverttubes.ResuspendallDNApelletsin30ulTE,incubatingatroomtemperatureor4oCforseveralhours.PREPARATIONOFSAMPLESFORSOUTHERNANALYSIS.Remove15ulofeachsampletoafreshtube.Add1ulDNase-freeRNase(BoehringerMannheim,1:1000dilutionof2U/ulstock)to1T,1Ts,andallSUPERNATANTfraction.ThePELLETfractionsdonothavetobe"RNased".Incubateatroomtemperatureforatleast30min.Addsamplebuffertoalltubes.Loadsamplesontoa0.6%agarosegelcontainingEtBrat0.2ug/ml.Includeasmallamountofsupercoiledtestplasmid(e.g.pDK370)intheDNAsizestandardstoserveasapositivecontrolforhybridization.Thefinalconcentrationofsupercoiledplasmidshouldbesuchthat~0.1ngisloaded.Rungelat20or30Vovernight.Photographgel.NotewhethertheintensitiesoftheøX174bandsareeveninalllanesandwhether2ucircleDNAisvisibleintheSUPERNATANTfractions.ProcessgelfortransfertoGeneScreenPlusaccordingtothePosiblotprotocol.Transferforatleast90min.UVcrosslinkDNAtomembrane.Airdry.HYBRIDIZATION.Prehybridizeblotat65oCfor~3hinChurchbuffercontaining0.5mg/mldenaturesalmonspermDNA(usually14mlChurchbufferplus0.7ml10mg/mlSSDNAperblot).Addprobe105cpmsperlane)andhybridizeat65oCovernight(atleast18h).Washblot2xwithBuffer1,15mineachat65oCand2xwithBuffer2,15mineachat65oC.Monitortheblot--thelastwashmaynotbenecessary.NOTE:Usethe0.9kbSmaI/PstIURA3fragmentfrompDK377tovisualizepDK370orotherURA3-containingplasmids.Usethe1.8kbSalI/ClaILEU2fragmentfrompDK255tovisualizeYCp41orotherLEU2-containingplasmids.Usethe~2kbXhoI/SalIfragmentfromCV13tovisualizeendogenous2ucircleDNA.Note:makeuptheYWB,EBB+2Xassaultbufferfresheachexperiment.| YWBper10ml5mL2MSorbitol(ifNZarrested,add40uL1.5mg/mL0.336mL1MK2HPO4N2to4mLYWB)0.064mL1MKH2PO44.6mLdH2O | YWB,glycerol,PMSF5mL2MSorbitol0.336mL1MK2HPO40.064mL1MKH2PO40.5mL100%ultrapureglycerol0.05mL0.2MPMSF4.05mLdH2O |
| 1XEBB,glycerol,PMSF(5mL)1mL5XEBB0.25mL100%glycerol12.5uL0.2MPMSF3.75uLdH2O | 1XEBB,glycerol,DTT,BSA(10mL)2mL5XEBB0.5mLglycerol10uL100mg/mLBSA10uL0.1MDTT7.48mLdH2O |
| 1XEBB,DTT,NaCl,BSA5mL1mL5XEbb5uL0.1MDTT0.1mL5MNaCl50uL100mg/mLBSA0.25mLglycerol3.6mLdH2O2mLEBB0.2mLNaCl0.1BSA | |
| REAGENTS | |
| YWB:50mL2Msorbitol16.8mL0.2MK2HPO43.2mL0.2MKH2PO430mLH2O | 0.1MDTT |
| 0.2MPMSFinethanolorispropanol | Glusulase |
| 5XEBB:5mL1MMgCl25mLTRIS-HCl,pH7.40.1mL0.5MEDTA90mLH2O | 5MNaCl |
| 1mMtaxolinDMSO | 2XASSAULTBUFFER:5mL10%SDS,UltraPure5mL0.5MEDTA5mLHEPES-KOH,pH7.6[7.5w/NaOH]85mLH2O**2XA.B.canbefrozenin10mLaliquots.[3.5mLAssaultbuffer,0.5mLtRNA/øX174] |
| tRNA/øX174DNA:1mL1mg/mLtRNAinH2O0.1mL10ug/mLuncutøX174DNA | 5XBRB80100mLs5Xstock80mMPipespH6.8(KOH)10mL800mMPipespH6.8(KOH)1mMEGTA1ml100mMEGTA1mMMgCl20.1mL1MMgCl2 |
MINICHROMOSOME-MICROTUBULEBINDINGASSAYGROWTHOFCELLS.Grow100mLofcellstoOD600=0.7-0.8at23oC.Forgoodbindingactivityithasprovedimportanttomaintaincellsinlogphase.Normally,2dayspriortodayofexperiment,asinglemedium-sizedcologyispickedfromselectivemediumandinoculatedinto5mLYPS(or-URA,asIdo).Twoadditionaldilutionsaremadefromthis"neat"inoculum(e.g.1:10and1:25).Thegoalistohavelatelogphaseculturesthenextday(~4x107cells/mL).Thedaypriortoexperiment,makea1:10dilutionofthesuitable5mLovernight.DeterminetheOD600andcorrelatingcelldensityfromthechart.Setupthree100mLYPDculturesatthefollowingdensities:~0.7x105,1x105and1.5x105cells/mL.Harvestcellsintwo50mLconicaltubes,1.5-2.0Kfor8-10min.WashwithH2Oandconsolidatecellsinonetube.Re-spin.NOTE:Ifcellshavebeenarrestedwithnocodazoleorafactor,includetheinhibitorintheH2Owash.SPHEROPLASTING.Re-suspendcellsin4mLYWB(containing10mMbeta-mercaptoethanoland1mMPMSF).Lesitatroomtemperaturefor2-3min.Add100uLglusulase.Incubateat23oCwithgentleshakingtoformspheroplasts.Spheroplstingshouldbecompletewithinonehour;avoidincubationslongerthan1h,15min.NOTE:Ifcellshavebeenarrestedwithnocodaoleoralphafactor,includetheinhibitorintheYWB.TransferspheroplastedcellstoaTOMYtube.Spinat3.5Kfor7min.Re-suspendcellsin4mLYWB(containing1mMPMSFonly)bygentlypipettingupanddown,usinga1mLplasticpipet.Re-spin.Repeatwashprocess,foratotaloftwowashes.NOTE:Ifcellshavebeenarrestedwithnocodazoleoralphafactor,includetheinhibitorintheFIRSTWASHONLY.Gentlyreuspendcellsin2.5mL1XEBB(containing0.1mMDTTand0.5mMPMSF).Transfertohomogenizer.Allowcellsto"swell"for10minatroomtemperature,thenhomogenizecellswith5pestlestrokes(upanddownisonestroke).Avoidintroducingair.TransfertocleanTOMYtube.Add150uL5MNaCl(finalconcentration~0.3M).Incubateatroomtemperaturefor5min.Add5mL1XEBB-plus(containing0.1mMDTT,150ug/mLBSA,and0.5mMPMSF).Thus,finalconcentrationsin7.5mLlysateare0.1MNaCland100ug/mLBSA.Incubateatroomtemperaturefor45min.Thawmicrotubule"seeds"andputat37oCfor30min.Addtaxolto10uM(e.g.1uL0.13mMtaxolto12uLMTs)andincubateanadditional15min.Remove250uLsampleforTOTALMATERIAL(so-called"1T").Keepthistube(and1Ts,seebelow)atroomtemperature,soastobecomparabletotheothersamples.DivideremaininglysatebetweentwoTOMYtubes.CLARIFYLYSATES.SpinTOMYtubesat15Kfor20min.Poolsupernatantsina10mLconicaltubeandmixgently.Aliquotlysatetomicrofugetubes.Theminimumnumberoftubesis[1+thenumberofbindingreactionsplanned](usually0.7-1mLextractpertube).Spinmicrofugetubesat15Kfor20min.Remove250uLSTARTINGMATERIAL("1TS")toafreshtube.Remove500uLclarifiedlysatefromeachmicrofugetubetoafreshtube.Add5uL1mMtaxol(inDMSO)toeachtube-finalconcentrationis10uMtaxol.Mixgently.Forastandardtitrationofbindingactivity,addmicrotubulestoeachtubeinincreasingamount.Normally,0,0.2,0.5,1,2,and4uLFor0.2uLand0.5uL,makea10-folddilutionofMTsinBRB80/30%glycerolbuffer(containing10uMtaxol).Allowbindingtoproceedatroomtemperaturefor15min.PelletMTs(andassociatedminichromosomes)at15Kfor8min.ItshouldbepossibletoseethelargerMTpellets(usuallybluish-white).Remove250uLofeachsupernatanttofreshtube.ThesearetheSUPERNATANTFRACTIONS.AspirateallresidualliquidwithdrawnoutPasteurpipets.ResuspendMTpelletsin250uLof1XEBB(containing0.1mMDTT,100mMNaCl,and1mg/mlBSA,butNOPMSF!).ThesearethePELLETFRACTIONS.NOTE:Ultimately,PELLETswillbe2XconcentratedrelativetoSUPs.Add250uLof2XASSAULTbuffer(containingtRNAandøX174DNA)toallsamples(i.e.allsupsandpellets,aswellas1Tand1Ts).Removeproteinbyadding5mLofProteinaseKsolution(20mg/mLfromBoehringerMannheim)toeachtube.Incubatetubesat50oCfor~1-1.5h.(longerisbetter).AfterProteinaseKtreatment,precipitateDNAbyadding250uL6MNH4OAcand500uLisopropanol.Storeat-20oCovernight.SpindownDNAinTOMYat15K,30min.4oC.Carefullyaspirateordecantsupernatants,removingasmuchsupernatantaspossible.NOTE:The"PELLET"pelletsareoftensmall.WashallDNApelletswith100-200uL70%ethanol.Spinbriefly(~5min.)andaspiratetheethanolsupernatant.AllowDNApelletstoairdry.Donotinverttubes.ResuspendallDNApelletsin30uLTE,incubatingatroomtemperatureor4oCforseveralhours.PREPARATIONOFSAMPLESFORSOUTHERNANALYSIS.Remove15uLofeachsampletoafreshtube.Add1uLDNase-freeRnase(BoehringerMannheim,1:1000dilutionof2U/uLstock)to1T,1Ts,andallSUPERNATANTfractions.ThePELLETfractionsdonothavetobe"RNased".Incubateatroomtemperatureforatleast30min.Addsamplebuffertoalltubes.Loadsamplesontoa0.6%agarosegelcontainingEtBrat0.2ug/mL.Includeasmallamountofsupercoiledtestplasmid(e.g.pDK370)intheDNAsizestandardstoserveasapositivecontrolforhybridization.Thefinalconcentrationofsupercoiledplasmidshouldbesuchthat~0.1ngisloaded.Rungelat20or30Vovernight.Photographgel.NotewhethertheintensititesoftheøX174bandsareeveninalllanesandwhether2ucircleDNAisvisibleintheSUPERNATANT(hopefully)fractions.ProcessgelfortransfertoGeneScreenPlusaccordingtothePosiblotprotocol.Transferforatleast90min.UVcrosslinkDNAtomembrane.Airdry.HYBRIDIZATION.Prehybridizeblotat65oCfor~3h.inChurchbuffercontaining0.5mg/mLdenaturedsalmonspermDNA(usually14mLChurchbufferplus0.7mL10mg/mLSSDNAperblot).Addprobe(105cpmsperlane)andhybridizaeat65oCovernight(atleast18h.).Washblot2XwithBuffer1,15min.eachat65oCand2XwithBuffer2,15min.eachat65oC.Monitortheblot-thelastwashmaynotbenecessary.NOTE:Usethe0.9kbSma1/Pst1URA3fragmentfrompDK377tovisualizepDK370orotherURA3-containingplasmids.Usethe1.8kbSal1/Cla1LEU2fragmentfrompDK255tovisualizeYCp41orotherLEU2-containingplasminds.Usethe~2kbXho1/Sal1fragmentfromCV13tovisualizeendogenous2ucircleDNA.| YWBforspheroplasting:5mLYWB25uL0.2MPMSF3.5uLbeta-ME | YWBforwash:8mLYWB40uL0.2MPMSF |
| 1XEBB(tobreak):2mL5XEBB25uL0.2MPMSF10uL0.1MDTT8mLH2O | 1XEBB-plus(todilute):1mL5XEBB12.5uL0.2MPMSF5uL0.1MDTT75uL100mg/mlBSA4mLH2O |
| 1XEBB-doubleplus(forMTpellets):1mL5XEBB12.5uL0.2MPMSF5uL0.1MDTT50uL100mg/mlBSA100uL5MNaCl3.9mLH2O | 2XAssaultBuffer:XmL2XASSAULTBUFFER(X=0.25mLx#samples)0.1xXmLtRNA/øX174DNA |
| 3.75dH2O | 0.25EDTA |
| 0.25SDS | 0.25HEPES |
| 0.5tRNA+øX174 |
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