YEASTTWO-HYBRIDSCREENWITHLIBRARYANDBAIT(ThefollowingprotocolisforusewiththeLIBRARYtransformationonly)Day1:Growanovernightcultureofasinglecolonyofyeasttransformedwithbaitvectorin2.5mlofSD-TrpmediumDay21.Thefollowingmorningdilutetheovernightcultureinto50mlofYPAD,andgrow4hoursina30Cshaker,withvigorousshaking(250-300rpm)2.Transfertoa50mlFalconTubeandpe 查看更多
Agatep,R.,R.D.Kirkpatrick,D.L.Parchaliuk,R.A.Woods,andR.D.Gietz(1998)TransformationofSaccharomycescerevisiaebythelithiumacetate/single-strandedcarrierDNA/polyethyleneglycol(LiAc/ss-DNA/PEG)protocol.TechnicalTipsOnline(http://tto.trends.com).Forcompleteinstructionsonhowtodoatwohybridscreenseethefollowingreferences.1.Gietz,R.D.,B.Triggs-Raine,A.Robbi 查看更多
GSTFusionProteinPurificationfromYeast5mlovernightcultureofyourfavoriteyeastinyourfavoritemedium.Inoculate50mlandgrow30oCshakingO/NuntilOD600=0.8to1.2.ForSCDculturesuse1/500and1/1500dilutions.Optional:addalpha-factorto2.5 M.Continueshakingat30oCfor60min.Add1MNaN3to10mM(finalconcentration)andmoveculturestoice.Everythingmustremaincoldfromhereono 查看更多
GSTFusionProteinPrepItalicsindicateoptionalstepsespeciallyusefulfortheanalysisofuntestedproteins.GROWTHANDHARVESTINGOFBACTERIAAdd100ulampicillinto100mlLB.Inoculateandgrowovernight.Inthemorning,addovernightbrothto900mlprewarmedLB.Grow1hourortoA600greaterthan0.5.Ampicillincanbeaddedtohelpmaintaintheplasmid.Makeglycerolstockfromovernightcultureifneede 查看更多
HahnLabLastmodifiedTue,Oct6,1998AminoAcidMix(40X)Use0.6g/literTyrosine2gArginine4gSerine2gValine2gThreonine4gIsoleucine2gPhenylalanine2gAsparticAcid2gProline2gGlucoseCompletePlates(1liter):1.7gYeastNitrogenbase-aminoacidsandammoniumsulfate5.0gAmmoniumSulfate1literH2OpHto7.0usingNaOHAdd:0.6gAminoAcidmix(mixwellbeforeadding)20gBacto-agar(Difco)Autocl 查看更多
COMPLEXMEDIAYPADmediumorAlsoCalledYPDplusAdenineYeastExtract-Peptone-DextroseplusAdeninemedium6.0gmyeastextract(Difco)12.0gmPeptone(Difco)12.0gmGlucose60mgadeninehemisulphate600mldistilledwaterBacto-agar(Difco)10g(addedforagarmedium)Sterilisebyautoclavingat121°Cfor15min.Wealwaysusea1literflaskandmake600mlsofmediabecauseitgivesexactly20plates(1Slee 查看更多
100XYC(7DropoutAminoAcidLiquid)1.0garginine0.5gasparticacid0.5gisoleucine0.5gphenylalanine0.5gproline0.5gserine1.0gthreonine0.5gvalineinfinalvolumeof100mlstirwithlowheatuntilintosolutionfiltersterilze10XYNB+tyrosine17gYNB(w/oaminoacidsandw/oammoniumsulfate)50gammoniumsulfate0.5gtyrosineinafinalvolumeof1Lstirwithlowheatuntilintosolutionfiltersterilz 查看更多
YeastGenomicDNAIsolationDavidAmbergGrow100mlculturetoabout1x10tothe8thSpin4x15mlsofculturedownabout3kx3min.ResUSPendentirepoolinabout6mlsddH2O,aliqouteinto1.5mlmicrofugetubesandspindown.Aspirateoffsuperandvortexpellettillsuspended.Add200ulsolnA,add200ulPhenol/CHCl3and0.3gacidwashedglassbeads.Vortex2min,add200ulTEpH8andspinatmaxspeedfor5min.Transfer 查看更多
DNAisolationfromyeast1)Starta5mlo/nculture2)Pelleto/ncultureinapurplescrewcaptube(thismaytakeseveralspins),anddumps/n.3)Add100 lofSTETtopelletandvortexbriefly.4)Addaboutacapfullof0.45mmglassbeads(makesuretherearenobeadsinthecapthreadsandtightenwelltopreventleakage).5)Vortexfor5min.(8ifusingmultiheadvortex)atRT6)Addanother100 lofSTET,and 查看更多
YeastTransformationUsingFrozenCompetentCellsandSingle-strandedDNAasaCarrierKatherineKolorReferences:SchiestlandGietz(1989)Curr.Genetics16:339-346Gietz,etal(1995)Yeast11:355-360Preparingcompetentcells:1.Inoculate100mlyeastsyntheticcompletemedium+3%glycerolmediumfromovernightculture.Growcultureto0.5-1.0X107cells/ml.(2-3Xhigherefficiencyifdilutedbackt 查看更多
LindaHoskins/HahnLabLastmodifiedWed,Jul5,2000Thismoreinvolvedmethodgiveshighertransformationefficiencythanthesemirapidmethod.UseDMSOiftransformingalibrarytoyeastand/ortransformingamutantstrainwhichtransformspoorlyusingtheLiOAcmethod.1.Inoculate5mlsYPDwithasingleyeastcolony.GrowO/Nat30degreesC.2.Add0.5mlscultureto4.5mlsfreshmedia,checkA660.Addsuitab 查看更多
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