Stuffyouneed:10XLiAc1MLiAc,filtersterilize50%PEG3350,filtersterilize 1.Inoculate2-5mlsofliquidYPEDor10mlsyntheticmediaandincubatewithshakingovernightat30oC. 2.Counto/ncultureandinoculate50mlsofwarmYPEDtoacelldensityof5x106/mlculture. Note: i)Diluteovernightcultures10-1ormoreinwater. ii)Carefullyplace10µlofthecellsUSPensionbetweenthecoverslipandthebaseofhaemocytometer.Letthecellssettleontothehaemocytometergridforafewminutes.Thegridareaistypically1squaremillimeter,dividedinto25equal-sizedsquares,andthevolumemeasuredis10-4ml. ii)Countthenumberofcellsin5diagonalsquares iv)Calculatethecelltiterasfollows:cellscountedx5xdilutionfactorx1/volumemeasuredbythe25squaresofthehaemocytometer.239cellsx5x10(dilutionfactor)x1/10-4ml=1.2x108cells/ml. v)Saccharomycescerevisiaedividesbybuddingfromamothercell.Countbuddedcellsasasinglecells.Countcellswithequalbudsizesastwocellsthereisevidenceofadditionalbudsformingoneithercell. vi)YoumayalsouseOD660todeterminecelltiter,howevertherelationshipbetweencellnumberandODisstrainspecific. 3.Incubatethecultureat30oConashakerat200rpmuntilitsequivalentto2x107cells/ml.Thiswilltake3to5hours.Thisculturewillgivesufficientcellsfor10transformations. Note: i)Itisimportanttoallowthecellstocompleteatleasttwodivisions.ii)Transformationefficiencyremainsconstantfor3to4celldivisions. 4.Harvestthecultureinasterile50mlcentrifugetubeat1000xgfor5min. 5.Pouroffthemedium,resuspendthecellsin25mlofsterilewaterandcentrifugeagain. 6.Pouroffthewater,resuspendthecellsin1.0ml100mMLiAcandtransferthesuspensiontoa1.5mlmicrofugetube. 7.Pelletthecellsattopspeedfor15secandremovetheLiAcwithamicroPipette. 8.Resuspendthecellstoafinalvolumeof500µl(2x109cells/ml)--about400µlof100mMLiAc-- Note: Ifthecelltiterofthecultureisgreaterthan2x107cells/mlthevolumeoftheLiAcshouldbeincreasedtomaintainthetiterofthissuspensionat2x109cells/ml.Ifthetiterofthecultureislessthan2x107cells/mlthendecreasetheamountofLiAc. 9.BoilSS-DNAfor5min.andquicklychillinicewater. ****ItisnotnecessaryordesirabletoboilthecarrierDNAeverytime.Keepasmallaliquotinyourownfreezerboxandboilafter3-4freeze-thaws.Butkeeponicewhenout.**** 10.Vortexthecellsuspensionandpipette50µlsamplesintoµfugetubes.PelletthecellsandremovetheLiAcwithamicropipette. 11.Thebasic"transformationmix"consistsof: 240µlPEG(50%w/v)36µl1.0M.LiAc50µlSS-DNA(2.0mg/ml)XµlDNA34-XµlSterileddH2O360µlTOTAL Carefullyaddtheseingredientsintheorderlisted. Note: OnecanalsopremixtheingredientsexceptfortheDNAthenadd355µlofTRAFOmixontopofthecellpellet.Thenadd5µlofDNAandmix.TakecaretodeliverthecorrectvolumeastheTRAFOmixisviscous. 12.Vortexeachtubevigorouslyuntilthecellpellethasbeencompletelymixed.Usuallytakesabout1min. 13.Heatshockinawaterbathat42oCfor40min. Note: Theoptimumtimecanvaryfordifferentyeaststrains.Pleasetestthisifyouneedhighefficiencyfromyourtransformations. 14.Microfugeat6-8000rpmfor15secandremovethetransformationmixwithamicropipette. 15.Pipette600µlofsterilewaterintoeachtubeandresuspendthepelletbypipettingitupanddowngently. Note: BegentleaspossIBLeatthisstepifhighefficiencyisimportant. 16.Plate! Note: WhenspreADIngyeastinoculumontotheplategentlydistributethefluidcompletelywithasterileglassrodwithaminimumofstrokes.Allowthefluidtobetakenupbytheplatepriortoincubation. 17.Incubateplatesfor2-4daystorecovertransformants. PLATEsolution40%PEG33500.1MLiAc10mMTris-HCl,pH7.51mMEDTAHighefficiencyLiActransformation
ModifiedfromGietzHomepage.
Theorderisimportanthere!ThePEGshouldgoinfirst,whichshieldsthecellsfromthedetrimentaleffectsofthehighconcentrationofLiAc.
"LazyBones"transformation(plasmidtransformations)
TRANSFORMATION:PLATETransformation
AdaptedfromprotocolusedintheRinelab.NecessarySolutions:1XTELbuffer10mMTris-HCl,pH7.51mMEDTA0.1MLiAcPLATEsolution40%PEG33500.1MLiAc10mMTris-HCl,pH7.51mMEDTACOMPETENCY:Electroporation
1.Inoculate50mlYEPDwithacolonyandgrowwithshakingat30degCuntilearlystationary(~0.6-2E8cells/ml).2.Harvestina50mlsterileconicaltubeinGPRcentrifugespinat3000rpm,4degC,5"andkeepcellsonicethroughouttheprocedure.3.Washcellswith40mlICE-COLDsteriledH2O,pelletat2.5krpm,4degC,5".4.Repeatwashwith20mlsteriledH2O(ice-cold).5.Resuspendcellsin5ml1MSorbitol(ice-cold)pelletat2krpm,4degC,5".6.Resuspendcellswith150µl1MSorbitol(ice-cold)-KEEPONICE!7.Mix40µlofyeastsuspensionwith<5µlDNA(~5µg)inaprechilledelectroporationcuvette(0.2cm).Tapcontentstothebottom,makingsurethatthesampleisincontactwithbothsidesofthealuminumcuvette.8.Giveonepulse:V=1.5kV,25µF,200Ohms.Timeshouldbe~4-5".9.Immediatelyadd1ml1MSorbitol(ice-cold)andtransferwithasterilepasteurpipettetoasterileEppendorftube.10.Spreadontoselectiveplates.