TIP: i.CarrierDNAcanbefrozenafterboilingandused3or4times.IftransformationefficienciesbegintodecreasewithabatchofboiledcarrierDNAitshouldbeboiledagainoranewaliquotused.ii.ThelowerconcentrationofcarrierDNA(2mg/ml)inthisprotocoleaseshandlingandgivesmorereproducIBLeresults.iii.Inpreviousprotocolversions,aphenol:chloroformextractionwasusedtoensuremaximaltransformationefficiencies.ThisextractionmaynotbenecessaryiftheDNAisofhighenoughquality.TestyourcarrierDNAtodetermineifextractionisnecessary. Thelithiumacetatesolutionispreparedasa1.0Mstockindistilledde-ionizedwater(ddwater)andfiltersterilized.Thereisnoneedtotitratethissolution,yetthefinalpHshouldbebetween8.4-8.9. Thepolyethyleneglycol(PEG),MW3350(SigmaP3640)ismadeupto50%(w/v)withddwaterandfiltersterilized.Foroptimaltransformationefficiencies,caremustbetakentoensurethatthePEGsolutionisattheproperconcentration.Inaddition,itisimportanttostorethePEGinatightlycappedcontainertopreventevaporationofwaterandasubsequentincreaseinPEGconcentration.SmallvariationsaboveorbelowthePEGconcentrationoptimuminthetransformationreaction,whichis33%(w/v),canreducetheproductionoftransformants. *Note: EvaporationofthewaterfromthePEGstocksolutionwillresultinanincreaseintheeffectiveconcentrationofPEGinthetransformationreactionandseverelyreducetheefficiency. PlasmidDNAcanbepreparedbystandardprotocols;extensivepurificationisnotnecessaryforyeasttransformation.RNAisaneffectivecarrierintheLiAc/SS-DNA/PEGtransformationprocedure(SchiestlandGietz,1989)anddoesnotneedtoberemovedfromplasmidDNApreparationsbeforeTransformation.PleaseNote:YouDoNothavetoSonicatetheCarrierDNAANYMORE!WHY?IhavedonetheexperimentsandshownthatthatBiggerisBetterforCarrierDNA!Followinstructionbelow.
Single-strandedCarrierDNA(2mg/ml)
NoteonCarrierDNAsterility:WedissolveourcarrierDNAintosterileTEandthenconsideritsterileafterthe5mininaboilingwaterbath.Havenothadproblemswiththisapproach!
1.0MLithiumAcetateStockSolution
Note:LithiumAcetatecanbesterilizedintheautoclavefor20minsliquidcycle.
Polyethyleneglycol(PEG50%w/v)
NOTE:YOUCANALSOAUTOCLAVEPEGTOSTERILIZE!!!Itsabiteasier.
PlasmidDNA