Difference between revisions of "Yeast Transformation"
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*1M Lithium Acetate, autoclaved | *1M Lithium Acetate, autoclaved | ||
*Sterile water | *Sterile water | ||
| + | *Selective Agar Plates (see [[Yeast Selective Media and Agar]]) | ||
==Protocol== | ==Protocol== | ||
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#Prepare Transformation Mixture for the appropriate number of transformations: | #Prepare Transformation Mixture for the appropriate number of transformations: | ||
{| border="1" | {| border="1" | ||
| − | ! | + | ! Reagent || Concentration || 1 || 5(6X) || 10(11X) |
|- | |- | ||
|PEG 3500 || 50% || 240uL || 1440uL || 2640uL | |PEG 3500 || 50% || 240uL || 1440uL || 2640uL | ||
| Line 32: | Line 33: | ||
|Lithium Acetate || 1M || 36uL || 216uL || 396uL | |Lithium Acetate || 1M || 36uL || 216uL || 396uL | ||
|- | |- | ||
| − | |Herring Testes DNA || 10mg/mL || | + | |Herring Testes DNA || 10mg/mL || 10uL || 60uL || 110uL |
|- | |- | ||
|Water|| || 34uL || 204uL || 374uL | |Water|| || 34uL || 204uL || 374uL | ||
|- | |- | ||
| − | |Tube || eppendorf tube || 15mL Falcon Tube || 15 mL Falcon Tube | + | |Tube || || eppendorf tube || 15mL Falcon Tube || 15 mL Falcon Tube |
|} | |} | ||
Latest revision as of 17:14, 22 December 2009
Materials
Per strain, this is enough for 10 transformations per strain
- 50 mL autoclaved YPD in a 250 mL flask
- 200 mL 2xYPD
- Autoclaved empty 250 mL flask
- Herring testes DNA (Clontech Cat# 17401336)
- 50% PEG 3500, autoclaved
- 1M Lithium Acetate, autoclaved
- Sterile water
- Selective Agar Plates (see Yeast Selective Media and Agar)
Protocol
- Innoculate 50 mL YPD overnight in a 250 mL flask at 24C before going home
- The next morning check OD-600 of yeast.
- Add 10 uL cells to 1 mL of water and measure OD-600.
- Adjust for dilution factor (100X) and calculate OD.
- Dilute to a final OD of 0.5 in 50 mL of 2xYPD in an empty autoclaved 250 mL flask.
- Incubate at 24C for 2 divisions (typically ~4.5h).
- Centrifuge cells at 3000g for 5min.
- Boil hering tested DNA for 5 min at 95C then cool on ice.
- Gently resuspend in 25mL sterile water.
- Centrifuge cells at 3000g for 5min.
- Gently resuspend cells in 1mL sterile water and transfer to an eppendorf tube
- Centrifige 30s in a microfuge and aspirate supernatant
- Resuspend with 1mL sterile water ensuring to break up any clumps.
- Transfer 100 uL aliquots of cells to 1.5mL tubes, one for each transformation.
- Prepare Transformation Mixture for the appropriate number of transformations:
| Reagent | Concentration | 1 | 5(6X) | 10(11X) |
|---|---|---|---|---|
| PEG 3500 | 50% | 240uL | 1440uL | 2640uL |
| Lithium Acetate | 1M | 36uL | 216uL | 396uL |
| Herring Testes DNA | 10mg/mL | 10uL | 60uL | 110uL |
| Water | 34uL | 204uL | 374uL | |
| Tube | eppendorf tube | 15mL Falcon Tube | 15 mL Falcon Tube |
- Add 360 uL Transformation Mixture to each tube of cells and mix by vortexing.
- Incubate at 42C for 40min.
- Centrifuge for 30s and aspirate supernatant
- Add 1mL sterile water, resuspend and mix by gently vortexing.
- Plate 50 uL on appropriate selective plate.
- Allow to grow for 3-4 days at 24C.
Based on PMID 17401336. See http://home.cc.umanitoba.ca/~gietz/method.html
