YEASTTWO-HYBRIDSCREENWITHLIBRARYANDBAIT (ThefollowingprotocolisforusewiththeLIBRARYtransformationonly) Day1: Growanovernightcultureofasinglecolonyofyeasttransformedwithbaitvectorin2.5mlofSD-Trpmedium 1.Thefollowingmorningdilutetheovernightcultureinto50mlofYPAD,andgrow4hoursina30Cshaker,withvigorousshaking(250-300rpm) 2.Transfertoa50mlFalconTubeandpelletcells(10minat2500Kinaclinicalcentrifuge) 3.ResUSPendthepelletin1mlof0.1MLiOAc 4.TransfertoanEppendorftubeandspinattopspeedfor1mintopelletcells 5.Resuspendthepelletin500ulof0.1MLiOAc 6.Transformation: Aliquot100ulofcellsintoeachof3eppendorftubes,quickspin,andtakeoffsup.Thenaddoverthepellet,thefollowinginthefollowingorder: 500ulof50%PEG3350 10ulofboiledHerringspermDNA(placein100Cblockfor5min,thenplaceonicefor2min). 72ulof1MLiOAc 100ulofDNAasnotedbelow Tube1:Wateronly(noDNA) Tube2:1ugofpGAL4DNA Tube3:40ugofLibraryDNA 7.Vortextomix 8.30Cwaterbathfor30min,then 42Cwaterbathfor20min 9.Quickspintopellet,takeoffsup. 10.Resuspendpelletin1mlofYPAD 11.30-60minat30C 12.quickspin,takeoffsup 13.Resuspendinthefollowing: Tube1:400ulofwater Tube2:400ulofwater Tube3:3mlofwater 14.Plateonthefollowing: Tube1:200ulonasingleSMALLLeu/Trp/Hisplate 200ulonasingleSMALLTrp/Hisplate Tube2:400ulonasingleLARGELeu/Trp/Hisplate Tube3:400ulonasingleLARGELeu/Trpplate 400ulon7LARGELeu/Trp/Hisplates InvertandIncubateplatesfor2-3daysat30C.Day2