Day2

1.Determinethetiteroftheyeastculturebypipetting10mlofcellsinto1.0mlofwaterinaspectrophotometercuvetteandmeasuringtheODat600nm.FormanyyeaststrainsasUSPensioncontaining1x10⁶cells/mlwillgiveanOD600of0.1.Alternatively,titerthecultureusingahemocytometer.seenote:

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Note:

i)DiluteovernightYPADorSCcultures10^-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.Countcellswithequalbudsizesastwocellswhenthereisevidenceofadditionalbudsformingoneithercell.Somestrainsformclumpsofcellswhichreduceplatingefficiency.Asingleclumpofcellswillonlygiverisetoonecolonyonaplate,whichmaycomplicatefurtheranalysis.

---

2.Transfer50mlofthepre-warmed2xYPADtothepre-warmedcultureflaskandadd2.5x10⁸cellstogive5x10⁶cells/ml.

3.Incubatetheflaskonarotaryorreciprocatingshakerat30°Cand200rpm.

Note:

i)Itisimportanttoallowthecellstocompleteatleasttwodivisions.

ii)Thiswilltake3to5hours.

iii)Thisculturewillgivesufficientcellsfor10transformations.iv)Transformationefficiency(transformants/µgplasmid/10⁸cells)remainsconstantfor3to4celldivisions.

4.Whenthecelltiterisatleast2x10⁷cells/ml,whichshouldtakeabout4hours,harvestthecellsbycentrifugationat3000gfor5min,washthecellsin25mlofsterilewaterandresuspendin1mlofsterilewater.

5.Boila1.0mlsampleofcarrierDNAfor5minandchillinanice/waterbathwhileharvestingthecells.

****ItisnotnecessaryordesirabletoboilthecarrierDNAeverytime.Keepasmallaliquotinyourownfreezerboxandboilafter3-4freeze-thaws.Butkeeponicewhenout.****

6.Transferthecellsuspensiontoa1.5mlmicrocentrifugetube,centrifugefor30secanddiscardthesupernatant.

7.Addwatertoafinalvolumeof1.0mlandvortexmixvigorouslytoresuspendthecells.

Note:Ifthecelltiterofthecultureisgreaterthan2x10⁷cells/mlthevolumethenincreasethevolumetomaintainthetiterofthissuspensionat2x10⁹cells/ml.Ifthetiterofthecultureislessthan2x10⁷cells/mlthendecreasevolume.

8.Pipette100µlsamples(ca.10⁸cells)into1.5mlmicrofugetubes,oneforeachtransformation,centrifugeattopspeedfor30secandremovethesupernatant.

9.MakeupsufficientTransformationMixfortheplannednumberoftransformationsplusoneextra.KeeptheTransformationMixinice/water.

10.Add360µlofTransformationMixtoeachtransformationtubeandresuspendthecellsbyvortexmixingvigorously.

11.Incubatethetubesina42°Cwaterbathfor40min.

Note:Theoptimumtimecanvaryfordifferentyeaststrains.Pleasetestthisifyouneedhighefficiencyfromyourtransformations.

12.Microcentrifugeattopspeedfor30secandremovetheTransformationMixwithamicropipettor.

13.Pipette1.0mlofsterilewaterintoeachtube;stirthepelletbywithamicropipettetipandvortex.

Note:WeliketobeagentleaspossIBLeatthisstepifhighefficiencyisimportant.Excessivewashingwashesawaytransformants!!!!

14.PlateappropriatedilutionsofthecellsuspensionontoSCselectionmedium.Fortransformationwithanintegratingplasmid(YIp),linearconstructoroligonucleotide,plate200µlontoeachof5plates;foraYEp,YRporYCplibraryplasmiddilute10µlofthesuspensioninto1.0µlofwaterandplate10and100µlsamplesontotwoplateseach.The10µlsamplesshouldbepipetteddirectlyinto100µlpuddlesofsterilewaterontheSCselectionmedium.

Note:WhenspreADIngyeastinoculumontotheplategentlydistributethefluidcompletelywithasterileglassrodwithaminimumofstrokes.Allowthefluidtobetakenupbytheplatepriortoincubation.

15.Incubatetheplatesat30°Cfor3to4daysandcountthenumberoftransformants.Thetransformationefficiency(transformants/1µgplasmid/10⁸cells)canbedeterminedbycalculatingthenumberoftransformantsin1.0mlofresuspendedcellsper1.0microgramplasmidper10⁸cells.Forexample,ifthetransformationof1.0x10⁸cellswith100nanogramplasmidresultedin500coloniesonaplateofSCdropoutmediumspreadwith1µlofsuspension(usuallydispensedintoa100µlpuddleofsterilewaterontheplate).TransformationEfficiency=500x1000(platingfactor)x10(plasmidfactor)x1(cells/transformationx10⁸).TransformationEfficiency=5x10⁶transformants/1.0µgplasmid/10⁸cells.Transformationefficiencydeclinesasplasmidconcentrationisincreased(Gietzetal.1995)buttheactualyieldoftransformantspertransformationincreases.Forexample,100nanogramofplasmidinatransformationmightgiveaTransformationEfficiencyof5x10⁶andayieldof5x10⁵transformantswhereaswith1µgofplasmidtheTransformationEfficiencymightbe2x10⁶andtheyield2x10⁶pertransformation.Inordertoobtain5x10⁶transformantsitissimplertosetuptwoorthreetransformationswith1µgofplasmidDNA,orasingle3foldscaleduptransformation,thantocarryout10reactionswith100ngofplasmidineach.

Note:Twoplasmids,suchasanexpressionplasmidandalibraryplasmidpool,canbeco-transformedintoasinglecellbyincludingbothplasmidsinthesametransformationreaction.Theefficiencyisreduced,however,becauseonlyabout30-40%ofalltransformedcellstakeupmorethanoneplasmidmolecule.Analternativeapproach,whichhasahigherefficiencyistotransformintheexpressionplasmidfirst,usingthisortheQuickandEasyprotocol,andthenusetheprotocolfoundonthe2HybridSystemTRAFOPagetotransforminthelibraryplasmidpool.

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/**/

NumberofTransformations
Reagents	1	5(6X)	10(11X)
PEG350050%w/v	240µl	1440µl	2640µl
LiAc1.0M	36µl	216µl	396µl
BoiledSS-carrierDNA	50µl	300µl	550µl
PlasmidDNAplusWater	34µl	204µl	374µl
Total	360µl	2160µl	3960µl