Team:Calgary/Notebook/Protocols/Process6

From 2011.igem.org

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<li>Algal f2 media</li>
<li>Algal f2 media</li>
<li>Glass beads (0.45-0.52mm diameter)</li>
<li>Glass beads (0.45-0.52mm diameter)</li>
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<li>vector DNA</li>
<li>vector DNA</li>
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<p>Protocol</p>
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Revision as of 23:42, 27 September 2011


Plasmid Extraction

Reagents and Materials

  • Algal f2 media
  • Glass beads (0.45-0.52mm diameter)
  • Conc. sulfuric acid
  • ddH2O
  • 20% (w/v) PEG solution
  • D. salina culutre (log phase)
  • vector DNA

    • Protocol

    1. A solution containing 20% (w/v) PEG was prepared and then added to cells of D. salina before transformation.
    2. Glass beads, 0.45– 0.52 mm in diameter, were washed with concentrated sulfuric acid, then rinsed thoroughly with distilled water, and dried by baking at 180°C for 2–3 h.
    3. D. salina cells were cultured to logarithmic phase and then harvested by centrifugation at 5,000 rpm for 2 min.
    4. Cells were washed three times with the liquid medium as mentioned above, and resuspended with this medium at a concentration of 105 cells/ml.
    5. A mixture with 300 mg glass beads, 40 µg vector DNA (0.4 µg/µl) and 100 µl PEG was added to 0.8 ml of D. salina cell culture, mixed briefly by gently inverting tube and then agitated at 2,000 rpm on a vortex for 6 sec in 1.5 ml centrifuge tubes.
    6. The glass beads were allowed to settle, and cells were transferred to sterilized test tubes and cultured in liquid medium under dim light condition for 24 h.

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