Difference between revisions of "Yeast Transfection (Small Scale)"

Revision as of 15:01, 14 December 2009

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 30C 2. Transfer overnight culture to 300 mL YPDA to a OD600 of 0.2-0.3 3. Incubate 3h at 30C to an OD of 0.4-0.6 4. 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 5min 6. Discard supernatant and resuspend in 50 mL sterile TE or water 7. Centrifuge 1000g for 5min 8. Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations 9. 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 100 uL yeast cells and mix by vortexing 11. Add 0.6 mL PEG/LiAc solution and vortex to mix 12. Incubate at 30C for 30min with shaking 13. Add 70 uL DMSO 14. Heat shock for 15min at 42C swirling occasionally to mix 15. Chill on ice for 1-2 min 16. Centrifuge at high speed for 5 s to pellet cells 17. Resuspend in 0.5mL TE 18. Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media 19. Allow cells to grow for 3-5 days on selective media.