HighEfficiencyTransformationProtocol Thisprotocolcanbeusedtogeneratesufficienttransformantsinasinglereactiontoscreenmultipleyeastgenomeequivalentsforplasmidsthatcomplementaspecificmutation.Itcanalsobeusedtotransformintegratingplasmids,DNAfragmentsandoligonucleotidesforyeastgenomemanipulation.Finally,itisusedtooptimisetheconditionsfortransformationofaparticularyeaststrain,forexample,thetransformationofaplasmidlibraryintoatwo-hybridyeaststraintransformedwithabaitplasmidbytheRapidTransformationProtocol.TheHighEfficiencyProtocolcanalsobeemployedtotransformayeaststrainsimultaneouslywithtwodifferentplasmids,suchasthetwo-hybridbaitandpreyplasmids. Day1 Inoculatetheyeaststraininto5mlofliquidmedium(2xYPADorSCselectionmedium)andincubateovernightonarotaryshakerat200rpmand30°C.PlaceabottleofdoublestrengthYPADbroth(2xYPAD)anda250mlcultureflaskintheincubatoraswell. Day2 1.Determinethetiteroftheyeastculturebypipetting10mlofcellsinto1.0mlofwaterinaspectrophotometercuvetteandmeasuringtheODat600nm.FormanyyeaststrainsasUSPensioncontaining1x106cells/mlwillgiveanOD600of0.1.Alternatively,titerthecultureusingahemocytometer.seenote: i)DiluteovernightYPADorSCcultures10-1ormoreinwater. ii)Carefullyplace10µlofthecellsuspensionbetweenthecoverslipandthebaseofhaemocytometer.Letthecellssettleontothehaemocytometergridforafewminutes.Thegridareaistypically1squaremillimeter,dividedinto25equal-sizedsquares,andthevolumemeasuredis10-4ml. ii)Countthenumberofcellsin5diagonalsquares iv)Calculatethecelltiterasfollows:cellscountedx5xdilutionfactorx1/volumemeasuredbythe25squaresofthehaemocytometer.239cellsx5x10(dilutionfactor)x1/10-4ml=1.2x108cells/ml. v)Saccharomycescerevisiaedividesbybuddingfromamothercell.Countbuddedcellsasasinglecells.Countcellswithequalbudsizesastwocellswhenthereisevidenceofadditionalbudsformingoneithercell.Somestrainsformclumpsofcellswhichreduceplatingefficiency.Asingleclumpofcellswillonlygiverisetoonecolonyonaplate,whichmaycomplicatefurtheranalysis. 2.Transfer50mlofthepre-warmed2xYPADtothepre-warmedcultureflaskandadd2.5x108cellstogive5x106cells/ml. 3.Incubatetheflaskonarotaryorreciprocatingshakerat30°Cand200rpm. Note: 4.Whenthecelltiterisatleast2x107cells/ml,whichshouldtakeabout4hours,harvestthecellsbycentrifugationat3000gfor5min,washthecellsin25mlofsterilewaterandresuspendin1mlofsterilewater. 5.Boila1.0mlsampleofcarrierDNAfor5minandchillinanice/waterbathwhileharvestingthecells. ****ItisnotnecessaryordesirabletoboilthecarrierDNAeverytime.Keepasmallaliquotinyourownfreezerboxandboilafter3-4freeze-thaws.Butkeeponicewhenout.**** 6.Transferthecellsuspensiontoa1.5mlmicrocentrifugetube,centrifugefor30secanddiscardthesupernatant. 7.Addwatertoafinalvolumeof1.0mlandvortexmixvigorouslytoresuspendthecells. Note:Ifthecelltiterofthecultureisgreaterthan2x107cells/mlthevolumethenincreasethevolumetomaintainthetiterofthissuspensionat2x109cells/ml.Ifthetiterofthecultureislessthan2x107cells/mlthendecreasevolume. 8.Pipette100µlsamples(ca.108cells)into1.5mlmicrofugetubes,oneforeachtransformation,centrifugeattopspeedfor30secandremovethesupernatant. 9.MakeupsufficientTransformationMixfortheplannednumberoftransformationsplusoneextra.KeeptheTransformationMixinice/water. NumberofTransformations 240µl 36µl 50µl 34µl 360µl 10.Add360µlofTransformationMixtoeachtransformationtubeandresuspendthecellsbyvortexmixingvigorously. 11.Incubatethetubesina42°Cwaterbathfor40min. Note:Theoptimumtimecanvaryfordifferentyeaststrains.Pleasetestthisifyouneedhighefficiencyfromyourtransformations. 12.Microcentrifugeattopspeedfor30secandremovetheTransformationMixwithamicropipettor. 13.Pipette1.0mlofsterilewaterintoeachtube;stirthepelletbywithamicropipettetipandvortex. Note:WeliketobeagentleaspossIBLeatthisstepifhighefficiencyisimportant.Excessivewashingwashesawaytransformants!!!! 14.PlateappropriatedilutionsofthecellsuspensionontoSCselectionmedium.Fortransformationwithanintegratingplasmid(YIp),linearconstructoroligonucleotide,plate200µlontoeachof5plates;foraYEp,YRporYCplibraryplasmiddilute10µlofthesuspensioninto1.0µlofwaterandplate10and100µlsamplesontotwoplateseach.The10µlsamplesshouldbepipetteddirectlyinto100µlpuddlesofsterilewaterontheSCselectionmedium. Note:WhenspreADIngyeastinoculumontotheplategentlydistributethefluidcompletelywithasterileglassrodwithaminimumofstrokes.Allowthefluidtobetakenupbytheplatepriortoincubation. 15.Incubatetheplatesat30°Cfor3to4daysandcountthenumberoftransformants.Thetransformationefficiency(transformants/1µgplasmid/108cells)canbedeterminedbycalculatingthenumberoftransformantsin1.0mlofresuspendedcellsper1.0microgramplasmidper108cells.Forexample,ifthetransformationof1.0x108cellswith100nanogramplasmidresultedin500coloniesonaplateofSCdropoutmediumspreadwith1µlofsuspension(usuallydispensedintoa100µlpuddleofsterilewaterontheplate).TransformationEfficiency=500x1000(platingfactor)x10(plasmidfactor)x1(cells/transformationx108).TransformationEfficiency=5x106transformants/1.0µgplasmid/108cells.Transformationefficiencydeclinesasplasmidconcentrationisincreased(Gietzetal.1995)buttheactualyieldoftransformantspertransformationincreases.Forexample,100nanogramofplasmidinatransformationmightgiveaTransformationEfficiencyof5x106andayieldof5x105transformantswhereaswith1µgofplasmidtheTransformationEfficiencymightbe2x106andtheyield2x106pertransformation.Inordertoobtain5x106transformantsitissimplertosetuptwoorthreetransformationswith1µgofplasmidDNA,orasingle3foldscaleduptransformation,thantocarryout10reactionswith100ngofplasmidineach. Note:Twoplasmids,suchasanexpressionplasmidandalibraryplasmidpool,canbeco-transformedintoasinglecellbyincludingbothplasmidsinthesametransformationreaction.Theefficiencyisreduced,however,becauseonlyabout30-40%ofalltransformedcellstakeupmorethanoneplasmidmolecule.Analternativeapproach,whichhasahigherefficiencyistotransformintheexpressionplasmidfirst,usingthisortheQuickandEasyprotocol,andthenusetheprotocolfoundonthe2HybridSystemTRAFOPagetotransforminthelibraryplasmidpool.
Note:Reagents PEG350050%w/v 1440µl 2640µl LiAc1.0M 216µl 396µl BoiledSS-carrierDNA 300µl 550µl PlasmidDNAplusWater 204µl 374µl 2160µl 3960µl