Bridges Lab Protocols

Yeast Transfection (Small Scale): Difference between revisions

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Materials
==Materials==
• YPDA Media 50 mL in a 250 mL flask and 300 mL in a 1L flask
*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
*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
*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
*Herring testes carrier DNA.  Boil 20min then place on ice before use
Protocol
==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
#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
#ransfer 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
#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.
#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
#Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min
6. Discard supernatant and resuspend in 50 mL sterile TE or water
#Discard supernatant and resuspend in 50 mL sterile TE or water
7. Centrifuge 1000g for 5min
#Centrifuge 1000g for 5min
8. Discard supernatant and resuspend in 1.5 mL of TE/LiAc.  This is enough for about 14 transformations
#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)
#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
#dd 100 uL yeast cells and mix by vortexing
11. Add 0.6 mL PEG/LiAc solution and vortex to mix
#Add 0.6 mL PEG/LiAc solution and vortex to mix
12. Incubate at 30C for 30min with shaking
#Incubate at 30C for 30min with shaking
13. Add 70 uL DMSO
#Add 70 uL DMSO
14. Heat shock for 15min at 42C swirling occasionally to mix
#Heat shock for 15min at 42C swirling occasionally to mix
15. Chill on ice for 1-2 min
#Chill on ice for 1-2 min
16. Centrifuge at high speed for 5 s to pellet cells
#Centrifuge at high speed for 5 s to pellet cells
17. Resuspend in 0.5mL TE
#Resuspend in 0.5mL TE
18. Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media
#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.
#Allow cells to grow for 3-5 days on selective media.


[[Category:Yeast]]
[[Category:Yeast]]
[[Category:Molecular Biology]]
[[Category:Molecular Biology]]
[[Category:Transfection]]
[[Category:Transfection]]
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php/Yeast_Transfection_(Small_Scale)"