PleaseNote:YouDoNothavetoSonicatetheCarrierDNAANYMORE!WHY?IhavedonetheexperimentsandshownthatthatBiggerisBetterforCarrierDNA!Followinstructionbelow.

Single-strandedCarrierDNA(2mg/ml)

1. Weighout200mgsofhighmolecularweightDNA(DeoxyribonucleicacidSodiumSaltTypeIIIfromSalmonTestes,SigmaD1626)into100mlofTEbuffer(10mMTris-HClpH8.0,1.0mMEDTA).DispersetheDNAintosolutionbydrawingitupanddownrepeatedlyina10mlpipet.Mixvigorouslyonamagneticstirrerfor2-3hoursoruntilfullydissolved.Ifconvenient,leavethecoveredsolutionmixingatthisstageovernightinacoldroom.
2. AliquottheDNAandstoreina-20^oCfreezer.
3. Priortouse,analiquotshouldbeplacedinaboilingwaterbathforatleast5minandquicklycooledinanicewaterslurry.

   TIP:

   i.CarrierDNAcanbefrozenafterboilingandused3or4times.IftransformationefficienciesbegintodecreasewithabatchofboiledcarrierDNAitshouldbeboiledagainoranewaliquotused.ii.ThelowerconcentrationofcarrierDNA(2mg/ml)inthisprotocoleaseshandlingandgivesmorereproducIBLeresults.iii.Inpreviousprotocolversions,aphenol:chloroformextractionwasusedtoensuremaximaltransformationefficiencies.ThisextractionmaynotbenecessaryiftheDNAisofhighenoughquality.TestyourcarrierDNAtodetermineifextractionisnecessary.

   NoteonCarrierDNAsterility:WedissolveourcarrierDNAintosterileTEandthenconsideritsterileafterthe5mininaboilingwaterbath.Havenothadproblemswiththisapproach!

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   1.0MLithiumAcetateStockSolution

   Thelithiumacetatesolutionispreparedasa1.0Mstockindistilledde-ionizedwater(ddwater)andfiltersterilized.Thereisnoneedtotitratethissolution,yetthefinalpHshouldbebetween8.4-8.9.

   Note:LithiumAcetatecanbesterilizedintheautoclavefor20minsliquidcycle.

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   Polyethyleneglycol(PEG50%w/v)

   Thepolyethyleneglycol(PEG),MW3350(SigmaP3640)ismadeupto50%(w/v)withddwaterandfiltersterilized.Foroptimaltransformationefficiencies,caremustbetakentoensurethatthePEGsolutionisattheproperconcentration.Inaddition,itisimportanttostorethePEGinatightlycappedcontainertopreventevaporationofwaterandasubsequentincreaseinPEGconcentration.SmallvariationsaboveorbelowthePEGconcentrationoptimuminthetransformationreaction,whichis33%(w/v),canreducetheproductionoftransformants.

   1. Place50gmofpolyethyleneglycol,MW3350(Sigma)ina150mlglassbeakerandadd35mlsofddH20.
   2. Stirwithamagneticstirringbaruntildissolved.Thiswilltakeabout30min.
   3. Transferalloftheliquidtoa100mlgraduatedcylinder.Rinsethebeakerwithasmallamountofdistilledwater,addthistothegraduatedcylindercontainingthePEGsolution,andbringthevolumetoexactly100mls.Mixwellbyinversion.
   4. Filtersterilizeusinga0.45µmfilterunit(Nalgene),andstoreinasecurelycappedbottle.

   NOTE:YOUCANALSOAUTOCLAVEPEGTOSTERILIZE!!!Itsabiteasier.

   *Note:

   EvaporationofthewaterfromthePEGstocksolutionwillresultinanincreaseintheeffectiveconcentrationofPEGinthetransformationreactionandseverelyreducetheefficiency.

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   PlasmidDNA

   PlasmidDNAcanbepreparedbystandardprotocols;extensivepurificationisnotnecessaryforyeasttransformation.RNAisaneffectivecarrierintheLiAc/SS-DNA/PEGtransformationprocedure(SchiestlandGietz,1989)anddoesnotneedtoberemovedfromplasmidDNApreparationsbeforeTransformation.

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