Team:Tuebingen/Notebook/Protocols/yeast-trafo

From 2013.igem.org

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   <li>Scrape some yeast cells off a fresh <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/ypd">YPD plate</a> and inoculate in <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/ypd">liquid YPD</a> (= culture).</li>
   <li>Scrape some yeast cells off a fresh <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/ypd">YPD plate</a> and inoculate in <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/ypd">liquid YPD</a> (= culture).</li>
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   <li>Thaw ssDNA ("helper DNA" e.g. salmon sperm DNA).</li>
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   <li>Thaw ssDNA ("helper DNA" e.g. salmon sperm DNA) and heat for 10 min at 95 °C, then chill on ice.</li>
   <li>Pellet 1 mL of culture by centrifugation at > 13.000 rpm.</li>
   <li>Pellet 1 mL of culture by centrifugation at > 13.000 rpm.</li>
   <li>Discard supernatant and resuspend cells in 100 µL <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/onestep">ONE-STEP buffer</a>. Vortex heavily.</li>
   <li>Discard supernatant and resuspend cells in 100 µL <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/onestep">ONE-STEP buffer</a>. Vortex heavily.</li>

Revision as of 03:47, 4 October 2013

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Yeast Transformation
Back to Protocols

 

Procedure

  1. Scrape some yeast cells off a fresh YPD plate and inoculate in liquid YPD (= culture).
  2. Thaw ssDNA ("helper DNA" e.g. salmon sperm DNA) and heat for 10 min at 95 °C, then chill on ice.
  3. Pellet 1 mL of culture by centrifugation at > 13.000 rpm.
  4. Discard supernatant and resuspend cells in 100 µL ONE-STEP buffer. Vortex heavily.
  5. 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.
  6. Add 1 mL YPD, mix and spin 10 sec at full speed. Discard supernatant.
  7. Resuspend cell pellet in 1000 µL YPD and plate 100 µL directly on appropriate selective plates.
  8. Colonies appear after 2 days of incubation at 30 °C.


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