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Team:Tuebingen/Notebook/Protocols/yeast-trafo
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| - | <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols" style="font-size: | + | <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols" style="font-size: 18px">Back to Protocols</a> |
| + | <p> </p> | ||
| + | |||
| + | <h3>Procedure</h3> | ||
| + | |||
| + | <ol> | ||
| + | <li>Scrape some yeast cells off of a fresh YPD plate and inoculate in liquid YPD (= culture).</li> | ||
| + | <li>Thaw ssDNA ("helper DNA).</li> | ||
| + | <li>Pellet 1 mL of culture by centrifugation at > 13.000 rpm.</li> | ||
| + | <li>Discard supernatant and resuspend cells in 100 µL ONE-STEP buffer. Vortex heavily.</li> | ||
| + | <li>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.</li> | ||
| + | <li>Add 1 mL YPD, mix and spin 10 sec at full speed. Discard supernatant.</li> | ||
| + | <li>Resuspend cell pellet in 1000 µL YPD and plate 100 µL directly on appropriate selective plates.</li> | ||
| + | <li>Colonies appear after 2 days of incubation at 30 °C.</li> | ||
| + | <li></li> | ||
| + | </ol> | ||
</div> | </div> | ||
| + | |||
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Revision as of 03:25, 4 October 2013
Yeast Transformation
Back to Protocols
Procedure
- Scrape some yeast cells off of a fresh YPD plate and inoculate in liquid YPD (= culture).
- Thaw ssDNA ("helper DNA).
- 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.
