Quick and Easy TRAFO Protocol
来自 : 蚂蚁淘
ThisProtocolallowsforTRAFOwithanyYeastcellsource
Referenece;Gietz,R.D.andR.A.Woods.(2002)TRANSFORMATIONOFYEASTBYTHELiac/SSCARRIERDNA/PEGMETHOD.MethodsinEnzymology350:87-96.Whenjustafewtransformantsaresufficient,suchasthetransformationofatestplasmidoraGAL4BDfusionplasmid,thefollowingprotocolcanbeused.ThisprocedureisveryflexIBLeandcanbeappliedtoyeastcellsfromanumberofdifferentsources;however,forbestresults,usecellsfromafreshlygrownplate.
AllsolutionsusedinthisprotocolaredescribedintheTRAFOSolutionsPage
RapidTransformationProtocol
Day11.Inoculatetheyeaststrainina2cm2patchontoYPADagar(YPD12supplementedwith100mgadeninehemisulphateperliter)andincubateovernightat30C.Alternatively,theyeaststraincanbeinoculatedinto5mlofliquidmedium(2xYPADorSCselectionmediumandincubatedonashakerat30Cand200rpm.
Day21.HeatatubeofcarrierDNAinaboilingwaterbathfor5minandthenchillinice/water.2.Scrapea50mlblobofyeastfromtheYPADplateandsUSPendthecellsin1mlofsterilewaterina1.5mlmicrocentrifugetube.Thesuspensionwillcontainabout5x108cells.Cellsgrownovernightin2xYPADbrothwillreachatiterbetween1and2x108/ml;thetiterinSCmediumwillbeabout5x107/ml.Harvest2mlofaYPADcultureand5mlofaSCculture.Note:cellsinlogphasegrowthonagarorinliquidmediumwilltransformwithhighefficiency.3.Pelletthecellsattopspeedinamicrocentrifugefor30secanddiscardthesupernatant.4.*AddthefollowingcomponentsoftheTransformationMixtothecellpelletintheorderlisted:| Component | Volume(µl) |
| 1.PEG350050%w/v | 240µl |
| 2.LiAc1.0M | 36µl |
| 3.BoiledSS-CarrierDNA(2mg/ml) | 50µl |
| 4.PlasmidDNA(0.1to1µg)pluswater | 34µl |
| TotalVolume | 360µlBesuretovortexmixthecarrierDNAbeforepipettingit.5.Incubatethetubeinawaterbathat42°Cfor40to60min.Manylaboratorystrainswillyieldupto1x105transformants/mgplasmidafter60minincubation.Extendingthetimeat42°Cto180minincreasestheyieldto>1x106/µgwithsomestrains.6.Microcentrifugeattopspeedfor30secandremovetheTransformationMixwithamicropipettor.7.Pipette1.0mlofsterilewaterintothetubeandresuspendthecellsbystirringwithamicropipettetipandthenvortexmixingvigorously.8.Pipette10and100µlsamplesontoplatesofappropriateSCselectionmedium,incubateat30°Cfor3-4daysandisolatetransformants.The10µlsamplesshouldbepipettedinto100µlpuddlesofsterilewater.Thisprotocolcanbeusedwithculturesthathavebeenstoredatroomtemperatureorinarefrigerator.Theyieldwillbereducedwitholderculturesbutwillgenerallybesufficienttoisolateanumberoftransformantsofthedesiredgenotype. |
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