2 Hybrid System TRAFO Protocol
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Agatep,R.,R.D.Kirkpatrick,D.L.Parchaliuk,R.A.Woods,andR.D.Gietz(1998)TransformationofSaccharomycescerevisiaebythelithiumacetate/single-strandedcarrierDNA/polyethyleneglycol(LiAc/ss-DNA/PEG)protocol.TechnicalTipsOnline(http://tto.trends.com).
Forcompleteinstructionsonhowtodoatwohybridscreenseethefollowingreferences.
1.Gietz,R.D.,B.Triggs-Raine,A.Robbins,K.C.Graham,andR.A.Woods(1997)Identificationofproteinsthatinteractwithaproteinofinterest:Applicationsoftheyeasttwo-hybridsystem.MolCellBiochem172:67-79.
2.Parchaliuk,D.L.,R.D.Kirkpatrick,R.Agatep,S.L.SimonandR.D.Gietz(1999)Yeasttwo-hybridsystemscreening.TechnicalTipsOnline(http://tto.trends.com)(accepted).notonlineyet
SEETRAFOREFs
ThisTRAFOProtocolisfor2Hybridsystemscreens(Wehavedoneover30andstillcounting)
HighEfficiencyTransformationofayeaststrainrequiringmaintenanceofaplasmid.
- InoculatetheyeaststraincontainingthefirstplasmidintotheappropriatevolumeoftheappropriateSC-omissionmediuminaflaskandincubateat30oCovernight.
ForeachdifferentScaleupusetheappropriatesizeofculture
TRAFOSCALE 10X 30X 60X CultureSize 25mls 50mls 100mls - Determinethecelltiterandcalculatethevolumeofcellsthatyields2.5x108cellsforeach50mlsofYPADcultureneeded
TRAFOSCALE 10X 30X 60X YPADcultureSize 50mls 150mls 300mls #ofCellsneeded 2.5x108 7.5x108 1.5x109 - Pourthisculturevolumeintoanappropriatesterilecentrifugetubeandpelletthecellsat3000xgfor5min.ResUSPendthecellpelletintheappropriatevolumeofpre-warmed(30oC)YPADandtransfertoanothersterilecultureflask.
TRAFOSCALE 10X 30X 60X YPADcultureSize 50mls 150mls 300mls - Incubateat30oCwhileshakingat200rpmfor3to4hrsuntilthecelltiterreaches2x107cells/ml.
- Harvestthecellsbycentrifugationat3000xgfor5min.
- Washthecellpelletviaresuspensionwith1/2volumeofsddwaterandcollectionbycentrifugationasabove.
- Resuspendthepelletintheappropriatevolumeof100mMsterileLiAcandtransfertoanappropriatecentrifugetube.Incubatefor15minat30oC.Pelletthecellsagainbycentrifugationandremovethesupernatant.
- Add,intheorderfromtoptobottom,thecomponentsofthetransformationmixlistedinthetablebelowtoaseperatetubeandmixthoroughlybyvortexing.Addthetransformationmixtothecellpelletandvortexvigorouslytoresuspendthecellpellet.AlternativelyyoumayalsomixallcomponentsbuttheplasmidDNAtogether.AddtheentirevolumeofthetransformationmixminustheplasmidDNAtothecellpelletandthenaddtheplasmidDNAandmix.ThiswillkeepyoufromloosinganyplasmidDNAwhentransferingtheviscousliquidofthetransformationmixontopofthecells.
TRAFOSCALE 10X 30X 60X 50%PEG 2.4ml 7.20ml 14.40ml 1.0MLiAc 360µl 1.08ml 2.16ml SS-DNA(2mg/ml) 500µl 1.50ml 3.00ml LibraryplasmidDNA Aµl Bµl Cµl sddWater 340-Aµl 1.02-Bml 2.04-Cml Pleasenote:Thevaluesforeachscaleupshouldbemultipliedfromthesinglereactionvolumes.Previouslythe60XscaleupvaluesfortheLiAc,SS-DNA,andPlasmidDNAwereNOTcorrect!(Theywere90Xscale,sorry)ThenumbersshownhereareNOWcorrect!Thankstothepersonthatcaughtmyerrorandsorrytoallofyoubattlingtogetgood2HSscreensdone.Inaddition,Pleasenotethatwearenowadding2Xtheamountofcarrierthaninpreviousversionsofthisthispage.TIP:
- Vigourouslyvortexthecellpelletuntilitistotallyresuspended,whichshouldtakeabout1min.Ifyouhaveproblemsgettingthepelletresusupendedletitsitfor5minandthenvortex!
- Incubatethetransformationmixat30oCfor30min.
- Heatshockat42oCfortimeindicatedbytablebelowwithmixingbyinversionfor15secafterevery5min.
TRAFOSCALE 10X 30X 60X HeatshockTime 30min 40min 45-60min TIP:
Heatshockoflargescaletransformationsrequiretheculturetubetobeinvertedseveraltimesevery5minutestoequilibratethetemperaturequicklyinthelargervolume.
- Collectthecellsbycentrifugationasabove.GentlyresuspendthecellpelletinanappropriatevolumeofsddwaterandplateontoSCommissionmedium.Forour30Xand60X2hybridscreensweplateonto100largeplates.(Yes!thatiscorrect!Awholecaseoflargeplates)Thisgivesbettertransformationandlibrarycoverage!Forthe10Xscreensweusedless.
TRAFOSCALE 10X 30X 60X ResuspensionVolume 10mls 40mls 40mls TIP:
Transformationsforthetwo-hybridsystemwhichusetheactivationoftheHIS3geneforgeneticselectioncanbeplateddirectlyontoSComissionmediumlackingTryptophan,Leucine,andHistidine(Trp,Leu,His).Thetotalnumberoftransformantsscreenedshouldbecalculatedbyplatingofasmallaliquot(1-2µl)ontoapairofSComissionmediumlackingTrp-Leuplates.
- Incubatetheplatesfor3-5daysat30oCoruntilcoloniesappear.ForsometwohybridScreenwewaitaslongas14days!
DiscussionItisimportanttooptimizetheamountoflibraryplasmidDNAforeachstandardtransformation.AsshowninTable1below,suchatestisdonebytransformingincreasingamountsoflibraryplasmidDNAinastandardtransformationreaction.Fromthisexperiment,onecanseethatitismoreproductivetodo10standardtransformationreactionswith1µgorplasmidDNA(orscaleupthetransformationreaction)thantodoonestandardtransformationusing10µgofplasmid.ThiswillnotonlyensureefficientuselibraryDNAbutwillalsoreducesthenumberofyeastcoloniescontainingmorethanonelibraryplasmid,whichcanmakesubsequentanalysisdifficult.
Co-transformationoflibraryplasmidDNAwithaGAL4BDplasmidisnotasefficientastransformationofthelibraryDNAintoayeaststrainalreadycontainingtheGAL4BDplasmid.Therefore,itisrecommendedthattheGAL4BDplasmidbetransformedfirstintotheyeaststrainusingtheQuick&Easyprotocolandfollowedbytransformationofthelibrarywithprotocolabove;wehavescreenedasmanyas5.2x107transformantsfromasinglescaleduptransformationreactioninatwo-hybridscreen(R.D.Gietz,unpublisheddata).IncasesweretheGAL4BDfusionplasmidaffectsthegrowthoftheyeaststrain,itmaybeadvisabletoco-transformtheGAL4BDfusionplasmidandthelibraryplasmidDNA.Inthesecases,carefulattentiontothelevelsofeachplasmidinthetransformationreactionisnecessaryfortheproductionofthehighestefficiency.
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i.Thestandardtransformationreactioncanbescaledupto120Xastandardtransformationreaction,howeverwerarelyneedtogotothisscale.
ii.UseaaplasticpipetratherthanaglasspipettotransferthePEGsolutionasitadherestothesurfaceofglasspipetsandhampersthedeliveryofanexactvolume.
iii.ThevolumeofsddwaterandplasmidDNAmaybeadjusted,however,thetotalvolumeofthesecomponentsmustremainconstant.
TRAFOSCALE 10X 30X 60X 100mMLiAc 3mls 3mls 6mls - Add,intheorderfromtoptobottom,thecomponentsofthetransformationmixlistedinthetablebelowtoaseperatetubeandmixthoroughlybyvortexing.Addthetransformationmixtothecellpelletandvortexvigorouslytoresuspendthecellpellet.AlternativelyyoumayalsomixallcomponentsbuttheplasmidDNAtogether.AddtheentirevolumeofthetransformationmixminustheplasmidDNAtothecellpelletandthenaddtheplasmidDNAandmix.ThiswillkeepyoufromloosinganyplasmidDNAwhentransferingtheviscousliquidofthetransformationmixontopofthecells.
Thetwo-hybridsystemandothersimilargeneticscreensinyeastinvolvetheuseoftwodifferentplasmidsinasingleyeastcell.OneplasmidoftencontainsaclonedgeneorDNAsequencewhiletheotherplasmidcontainsalibraryofgenomicorCDNA.Whilebothplasmidscanbeco-transformedintoasingleyeastcell,itisoftenmoreefficienttotransformthelibrarypoolintoastrainalreadycontainingthefirstplasmid(R.D.Gietz,unpublisheddata).
Toprepareaplasmid-carryingstrainforanadditionaltransformation,theinitialgrowthphaseshouldbeusingSComissionmediauntilthecelltiterreaches1-2x107cells/ml.Thiswillmaintainthefirstplasmidduringthisgrowthphase.ThecellscanthenbesubculturedinYPADmediumfortwodoublingswithoutsignificantplasmidloss,iftheplasmidhasnoeffectonyeastgrowth.Toproducehighlycompetentyeastcellscontainingaselectableplasmid,followtheprotocolbelow.TodeterminetheScaleupneededforthedesirednumberoftransformants,werecommendtestingone"slibraryDNAwiththeprotocolbelowtodeterminethenumberofTransformantsthatcanbeexpected.Thenselecttheconditionsthatgivea"good"Transformationyieldandthenscaleupto30X,60Xoreven120Xobtainthedesirednumberoftransformants.(seeTable1&Discussion).
AllsolutionsusedinthisprotocolaredescribedintheTRAFOSolutionsPage
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