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). 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). ForeachdifferentScaleupusetheappropriatesizeofcultureForcompleteinstructionsonhowtodoatwohybridscreenseethefollowingreferences.
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.
AllsolutionsusedinthisprotocolaredescribedintheTRAFOSolutionsPageTRAFOSCALE 10X 30X 60X CultureSize 25mls 50mls 100mls
TRAFOSCALE | 10X | 30X | 60X |
YPADcultureSize | 50mls | 150mls | 300mls |
#ofCellsneeded | 2.5x108 | 7.5x108 | 1.5x109 |
TRAFOSCALE | 10X | 30X | 60X |
YPADcultureSize | 50mls | 150mls | 300mls |
- 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.
i.Thestandardtransformationreactioncanbescaledupto120Xastandardtransformationreaction,howeverwerarelyneedtogotothisscale.
ii.UseaaplasticpipetratherthanaglasspipettotransferthePEGsolutionasitadherestothesurfaceofglasspipetsandhampersthedeliveryofanexactvolume.
iii.ThevolumeofsddwaterandplasmidDNAmaybeadjusted,however,thetotalvolumeofthesecomponentsmustremainconstant.
TRAFOSCALE | 10X | 30X | 60X |
100mMLiAc | 3mls | 3mls | 6mls |