1.MakeYPDgeneticinplates Geneticinisdissolvedinwaterataconcentrationof80mg(dryweight)permlandfilter-sterilized.Important!TheactiveconcentrationofGeneticiniswrittenonthebottleandvariesfromlottolot(from500to800µgpermgoftotaldryweight).Theamountusedperplatemustbeadjustedforeachlot.GibcoBRLreportsthatGeneticinisvirtuallyindestructable(itcanbeautoclaved--thoughthishasnotbeentried)andwehaveseenlittledecreaseinstrengthafterhavingplatessitatroomtempforamonth.Geneticinisaddedtofinalactiveconcentrationof300µg/mlto65°CYPDbrothcontaining2%Agar. 2.Dryplatesforseveraldaysatroomtemperature. (YPDisusedinsteadofYPADbecauseweuseacolonycolorassaytodetectsuccessfulAde2controltransformants.) 3.Start2mlliquidYPD(orYPAD)culturesofstrainstobeusedinthetransformation. Inthemorning,diluteovernightyeastcultures1:50in2mlsYPD(YPAD).Intheevening,(about8hourslater)checkO.D.600ofthecultures.HopefullytheO.D.sshouldbebetween1and5(notinstationaryphase).Inoculateaflaskcontaining250mlYPD(YPAD)withtheappropriatevolumeofcells.Thisvolumecanbecalculatedusingasimpleformula.Thisvolumewilldependonthedoublingtimeofthestrain,andthenumberofhoursbeforethetransformationwillbeperformed.ThetargetO.D.600fortheculturethenextmorningisabout1.5-2.0.250mlisenoughfor96transformations,whichwerarelydo.Moretypically,weperform36haploidand36diploidtransformations.Inthiscase100mlofcellsofeachisenough. 1.Prepare1Litersterile100mMLiAcetatesolutionfromsterile1Mstocksolution.2.PrepareLithiumAcetatePEGsolution: Mixexactly15grPEG-3350with16.5mlwater(thisisenoughfor96transformations).WhenPEGhasdissolved,filtersterilize.To12mlPEGsolutionAdd1.5mlsterilewater1.5ml1MLiAcetate 3.Turnon30°Cand42°Cwaterbaths.4.BoilcarrierDNAfor10minutesandthenplaceonice. 1.Preparecells CheckO.D.600ofcells.TheO.D.shouldbearound1.5-2.5.Culturesatlowerdensitiesgiveworsetransformationefficiencies,evenifyouusetwiceasmanycells(determinedbyKexinYu).Pelletcellsbycentrifugationatroomtemperature.Dumpoffsupernatant.Washcells2Xin0.5vol100`mMLiAcetateResUSPendcellsin1/100vol100mMLiAcetate.IfOD600ofculturewasnot1.0thenadjustfinalvolume.(IfO.D.600was0.8,not1.0,thenresuspend250mlscellsin2.0mls,not2.5mletc.)Placecellsinatroughandadd1/9volcarrierDNA(boiledfor10minutesandthenplacedonice.) 2.Place5µlPCRproductintoeachwellofamicrotiterplateandadd25µlofcarrierDNA/cellmixture--mixwellbypipettingupanddown.3.Incubate15minutesat30°C.4.Add150µlLiAcetatePEGsolution--Mixwellbypipettingupanddown!5.Incubate30minutesat30°C(timesofupto1.5hourshavebeenusedhere).6.Add17µlDMSOtoeachwell.7.Heatshockbyplacingthemicrotiterplateina42°Cwaterbathfor15minutes.8.Pelletcellsbycentrifugingthemicrotiterplate5minutesinalow-speedcentrifuge(weuseaspeedvac--novacuum).9.RemovePEGbytiltingtheplateandcarefullydrawingoffPEGwithamultichannelpipettor.Trynottodisturbthecellpellet,butsomecelllossisinevitable.10.Add200µlYPADtoeachwellandmixwell.11.Incubatemicrotiterplateat30°Conarotaryshakerfor~4hours~200rpm.Theplatecanbeattachedwithtapetoarotaryshakerinawarmroom.12.Plateentirecontentsofeachwellonageneticinplate. Arowofplates(usually12atatime)issetuponthebenchtopwithlidsajarandsomebeads(20orso)arespoonedintoeachplate.Theentirecontentsofamicrotiterwellisaddedtoeachplate.Thelidsareplacedontheplatesandtheplatesarestackedandthebeadsareswirledaroundtodistributethecells.Thenthebeadsaredumpedout.Thebeadsarecollectedforre-use(followingsterilization). Usingthismethodittakesus3-4hourstoperform96transformations(onestrainonly)andaboutoneandahalfhourstoplateoutthecells.Theprocedureappearstoworkrobustlyinourhandsandmanyvariations,suchaslongerincubationtimesatthedifferentsteps)havebeentriedwithlittlelossinefficiency.Pleasefeelfreetoexperimentyourselfandletusknowwhatyouthinkworksbetter.MultiwellTransformationProtocol
Labor-savingimplements
Chemicals
Protocol
Twodaysbeforehand:
Onedaybeforehad:
Onthedayofthetransformation:
96-welltransformation
Replicaplatingdoesnotseemtobenecessaryif300µg/mlGeneticinisused.Itisprobablynecessaryifalowerconcentrationisusedbecausethebackgroundincreases.