HighefficiencyLiActransformation

ModifiedfromGietzHomepage.

Stuffyouneed:10XLiAc1MLiAc,filtersterilize50%PEG3350,filtersterilize

1.Inoculate2-5mlsofliquidYPEDor10mlsyntheticmediaandincubatewithshakingovernightat30^oC.

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.

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3.Incubatethecultureat30^oConashakerat200rpmuntilitsequivalentto2x10⁷cells/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:

Theorderisimportanthere!ThePEGshouldgoinfirst,whichshieldsthecellsfromthedetrimentaleffectsofthehighconcentrationofLiAc.

OnecanalsopremixtheingredientsexceptfortheDNAthenadd355µlofTRAFOmixontopofthecellpellet.Thenadd5µlofDNAandmix.TakecaretodeliverthecorrectvolumeastheTRAFOmixisviscous.

12.Vortexeachtubevigorouslyuntilthecellpellethasbeencompletelymixed.Usuallytakesabout1min.

13.Heatshockinawaterbathat42^oCfor40min.

Note:

Theoptimumtimecanvaryfordifferentyeaststrains.Pleasetestthisifyouneedhighefficiencyfromyourtransformations.

14.Microfugeat6-8000rpmfor15secandremovethetransformationmixwithamicropipette.

15.Pipette600µlofsterilewaterintoeachtubeandresuspendthepelletbypipettingitupanddowngently.

Note:

BegentleaspossIBLeatthisstepifhighefficiencyisimportant.

16.Plate!

Note:

WhenspreADIngyeastinoculumontotheplategentlydistributethefluidcompletelywithasterileglassrodwithaminimumofstrokes.Allowthefluidtobetakenupbytheplatepriortoincubation.

17.Incubateplatesfor2-4daystorecovertransformants.

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\"LazyBones\"transformation(plasmidtransformations)

PLATEsolution40%PEG33500.1MLiAc10mMTris-HCl,pH7.51mMEDTA

• Take0.5mlofcultureandspin10secinmicrofuge.Decantthetubebyinvertingitandshakingitonce.Alternatively,onecanpickacolony(2-3mmindiameter)fromaplatewithatoothpickandtransfercellstosterile1.5mlmicrofugetube(aslongastheplateisnotdriedout,coloniescanbeusedfromplatesstoredinthefridgefor3months,maybemore).
• Add10µlofcarrierDNA(100µg)plus1µgtransformingDNA(in10µl)andvortexwell.(CarrierDNAdoesnotneedtobeaddedifthetransformingDNAhascomefrommini-prepDNAwhichhasnotbeenRNased).
• Add0.5mlPLATEsolutionandvortex.
• Add57µlDMSOandvortexbriefly.
• Leavefor15minatRT.
• Heatshockfor15minat42degC.
• Pelletcellsinmicrofugeforafewsecondsat10krpm.Carefullyremovesupernatant.
• Add200µlTEtothecellpelletandgentlyresuspendcellsbyaspiratingup-and-downwithapipettetip.Immediatelyspreadsuspendedcellsontoselectiveplates.NOTES-theyieldoftransformantsincreaseslinearlyuptoabout100-200µgoftransformingDNA.-theoptimalnumberofcellspertransformationisabout2E8cells/ml.Cells+DNAvolumeshouldbeabout140µl.Inotherwords,PLATE:(Cells+DNA)shouldbeabout3.5:1.

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  PLATETransformation

  AdaptedfromprotocolusedintheRinelab.NecessarySolutions:1XTELbuffer10mMTris-HCl,pH7.51mMEDTA0.1MLiAcPLATEsolution40%PEG33500.1MLiAc10mMTris-HCl,pH7.51mMEDTACOMPETENCY:
• Inoculateasinglecolonyinto100mlYPD(orselectivemediaifnecessary).
• GrowonshakertoO.D.(600)=1.0(O.D.(600)between0.6and1.8isfine)at30^oCovernight.
• Spindowncellsintabletopcentrifugeat2,000rpm.
• Resuspendcellsin10mlTEL.
• Shakevigorouslyovernightatroomtemperature;youcanskipthisstepandproceedtothenextifyoudon\"twanttosavecells.
• Spindowncellsandresuspendin1mlTEL.
• Usefortransformation.Cellscanbekeptintherefrigeratorandusedforuptoamonth.
TRANSFORMATION:
• Add50ugsalmonspermDNA(usually5ulof10mg/ml)to100ulofcompetentcellsinasterile1.5mltube.
• AddDNAtosuspension.Typically1ugofQiagenDNA,or5ulofminiprepDNAforuncutplasmids.Usemoreforintegratingconstructs.
• Incubate30minutesatroomtemperature.
• Add0.7mlPLATEsolution,resuspendthoroughly.
• Incubate1houratroomtemperature.
• Heatshockat42^oCfor5-10minutes.Plateonappropriateselectiveplates.

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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.