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Multiwell Transformation Protocol
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.
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