MultiwellTransformationProtocol

Labor-savingimplements

• Multichannelpipettors--1to25µland20to200µlvolume(Brinkmann12-channel).
• Sterile96-wellassayortissuecultureplatewithlid(suchasFalcon#1172).*Othersterileplates(flat,V,orUbottom)withwellvolumesof~300µlwillalsowork.
• Centrifugeforspinningmultiwellplates.
• Horizontalrotaryshaker,towhichamultiwellplatecanbeattached(bytape).
• Sterileglassbeads(5mmdiameter)+sterilespoon.
• Solutiontroughsformultichannelpipettors(labcor#730-004).

Chemicals

• PEG-3350(SigmaP3640)
• Lithiumacetatedihydrate(SigmaL-6883)
• Geneticin(GibcoBRL11811-031)
• 10mg/mlshearedsalmonspermDNA(BoehringerMannheim1467140)
• YPDbroth
• DMSO(Sigma)

Protocol

Twodaysbeforehand:

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.

Onedaybeforehad:

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.

Onthedayofthetransformation:

1.Prepare1Litersterile100mMLiAcetatesolutionfromsterile1Mstocksolution.2.PrepareLithiumAcetatePEGsolution:

Mixexactly15grPEG-3350with16.5mlwater(thisisenoughfor96transformations).WhenPEGhasdissolved,filtersterilize.To12mlPEGsolutionAdd1.5mlsterilewater1.5ml1MLiAcetate

3.Turnon30°Cand42°Cwaterbaths.4.BoilcarrierDNAfor10minutesandthenplaceonice.

96-welltransformation

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.

Replicaplatingdoesnotseemtobenecessaryif300µg/mlGeneticinisused.Itisprobablynecessaryifalowerconcentrationisusedbecausethebackgroundincreases.