YEASTTWO-HYBRIDSCREENWITHLIBRARYANDBAIT

(ThefollowingprotocolisforusewiththeLIBRARYtransformationonly)

Day1:

Growanovernightcultureofasinglecolonyofyeasttransformedwithbaitvectorin2.5mlofSD-Trpmedium

Day2

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.