Changes

==Materials==• *YPDA Media 50 mL in a 250 mL flask and 300 mL in a 1L flask• *TE/LiAc prepare fresh by combining 150 uL 10X TE, 150 uL 1M LiAc and 1.2 mL water• *PEG/LiAc prepare fresh by adding 8 mL 50% PEG-3350 to 1 mL 10X TE and 1 mL 1M LiAc in a 15 mL falcon tube• *Herring testes carrier DNA. Boil 20min then place on ice before use==Protocol==1. #Grow 50mL overnight culture of yeast in YPDA or SD. Add several colonies to 1mL vortexing to break up clumps then pouring into 50 mL media in a 250 mL Erlenmeyer flask. Grow at 30C2. Transfer #ransfer overnight culture to 300 mL YPDA to a OD600 of 0.2-0.33. #Incubate 3h at 30C to an OD of 0.4-0.64. #Place Herring DNA on heating block at 95C for 20min then immediately on ice before use.5. #Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min6. #Discard supernatant and resuspend in 50 mL sterile TE or water7. #Centrifuge 1000g for 5min8. #Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations9. #For each cotransformation combine 0.2 ug Bait (pGBT) and 0.1 ug Prey (pGADGH) with 0.1 mg Herring DNA (10 uL)10. Add #dd 100 uL yeast cells and mix by vortexing11. #Add 0.6 mL PEG/LiAc solution and vortex to mix12. #Incubate at 30C for 30min with shaking13. #Add 70 uL DMSO14. #Heat shock for 15min at 42C swirling occasionally to mix15. #Chill on ice for 1-2 min16. #Centrifuge at high speed for 5 s to pellet cells17. #Resuspend in 0.5mL TE18. #Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media19. #Allow cells to grow for 3-5 days on selective media.