Difference between revisions of "Yeast Transfection (Small Scale)"
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| − | Materials | + | ==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 | + | ==Protocol== |
| − | + | #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 | |
| − | + | #ransfer overnight culture to 300 mL YPDA to a OD600 of 0.2-0.3 | |
| − | + | #Incubate 3h at 30C to an OD of 0.4-0.6 | |
| − | + | #Place Herring DNA on heating block at 95C for 20min then immediately on ice before use. | |
| − | + | #Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min | |
| − | + | #Discard supernatant and resuspend in 50 mL sterile TE or water | |
| − | + | #Centrifuge 1000g for 5min | |
| − | + | #Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations | |
| − | + | #For each cotransformation combine 0.2 ug Bait (pGBT) and 0.1 ug Prey (pGADGH) with 0.1 mg Herring DNA (10 uL) | |
| − | + | #dd 100 uL yeast cells and mix by vortexing | |
| − | + | #Add 0.6 mL PEG/LiAc solution and vortex to mix | |
| − | + | #Incubate at 30C for 30min with shaking | |
| − | + | #Add 70 uL DMSO | |
| − | + | #Heat shock for 15min at 42C swirling occasionally to mix | |
| − | + | #Chill on ice for 1-2 min | |
| − | + | #Centrifuge at high speed for 5 s to pellet cells | |
| − | + | #Resuspend in 0.5mL TE | |
| − | + | #Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate 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]] | ||
Latest revision as of 15:02, 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
- 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
- ransfer overnight culture to 300 mL YPDA to a OD600 of 0.2-0.3
- Incubate 3h at 30C to an OD of 0.4-0.6
- Place Herring DNA on heating block at 95C for 20min then immediately on ice before use.
- Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min
- Discard supernatant and resuspend in 50 mL sterile TE or water
- Centrifuge 1000g for 5min
- Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations
- For each cotransformation combine 0.2 ug Bait (pGBT) and 0.1 ug Prey (pGADGH) with 0.1 mg Herring DNA (10 uL)
- dd 100 uL yeast cells and mix by vortexing
- Add 0.6 mL PEG/LiAc solution and vortex to mix
- Incubate at 30C for 30min with shaking
- Add 70 uL DMSO
- Heat shock for 15min at 42C swirling occasionally to mix
- Chill on ice for 1-2 min
- Centrifuge at high speed for 5 s to pellet cells
- Resuspend in 0.5mL TE
- Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media
- Allow cells to grow for 3-5 days on selective media.
