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Team:Tuebingen/Notebook/Protocols/yeast-trafo
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Latest revision as of 12:00, 4 October 2013
Yeast Transformation
Procedure
- Scrape some yeast cells off a fresh YPD plate and inoculate in liquid YPD (= culture).
- Thaw ssDNA ("helper DNA" e.g. salmon sperm DNA) and heat for 10 min at 95 °C, then chill on ice.
- Pellet 1 mL of culture by centrifugation at > 13.000 rpm.
- Discard supernatant and resuspend cells in 100 µL ONE-STEP buffer. Vortex heavily.
- Add 20 µg ssDNA (10 µL of 2 mg/mL) and 100 - 500 ng plasmid DNA to be transformed. Vortext and incubate at 45 °C for 2 h.
- Add 1 mL YPD, mix and spin 10 sec at full speed. Discard supernatant.
- Resuspend cell pellet in 1000 µL YPD and plate 100 µL directly on appropriate selective plates.
- Colonies appear after 2 days of incubation at 30 °C.
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