Team:Calgary/Notebook/Protocols/Process8

From 2011.igem.org

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TITLE=Nuclear DNA extraction from Algae|
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TITLE=Nuclear DNA Extraction from Algae|
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Total DNA isolation protocol
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<h4>Total DNA Isolation Protocol</h4>
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<ul>
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<p><h5>Reagents and Materials</h5></p>
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<p><i>Extraction buffer</i> (for 40 ml total):
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<table border="1px">
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    <tr>
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      <td>1 M Tris HCl pH 7.5</td>
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      <td>8 ml</td>
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    </tr>
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    <tr>
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      <td>5 M NaCl</td>
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      <td>2 ml</td>
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    </tr>
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    <tr>
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      <td>0.5 M EDTA</td>
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      <td>2 ml</td>
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    </tr>
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    <tr>
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      <td>20% SDS</td>
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      <td>1 ml</td>
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    </tr>
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    <tr>
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      <td>ddH<sub>2</sub>O</td>
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      <td>27 ml</td>
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    </tr>
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</table></p>
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<p><i>Elution buffer</i> (provided in kit from Qiagen)
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</p>
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<p><h5>Protocol</h5></p>
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<ol>
<li>Place 1.5 ml of algae culture into centrifuge tube.</li>
<li>Place 1.5 ml of algae culture into centrifuge tube.</li>
<li>Spin down cells at 5000 rpm for 1 minute.</li>
<li>Spin down cells at 5000 rpm for 1 minute.</li>
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<li>Centrifuge at 13000 rpm for 2 minutes.</li>
<li>Centrifuge at 13000 rpm for 2 minutes.</li>
<li>Discard the supernatant, DNA is in the pellet.</li>
<li>Discard the supernatant, DNA is in the pellet.</li>
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</ol>
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Latest revision as of 04:14, 29 September 2011


Nuclear DNA Extraction from Algae

Total DNA Isolation Protocol

Reagents and Materials

Extraction buffer (for 40 ml total):

1 M Tris HCl pH 7.5 8 ml
5 M NaCl 2 ml
0.5 M EDTA 2 ml
20% SDS 1 ml
ddH2O 27 ml

Elution buffer (provided in kit from Qiagen)

Protocol

  1. Place 1.5 ml of algae culture into centrifuge tube.
  2. Spin down cells at 5000 rpm for 1 minute.
  3. Pipette off supernatant.
  4. Add 400 µl extraction buffer.
  5. Incubate for 15 minutes at 50ºC, inverting tubes several times during incubation.
  6. Centrifuge at 13000 rpm for 5 minutes.
  7. Transfer supernatant to new tube.
  8. Add 400 µl isopropanol.
  9. Invert several times.
  10. Place on ice for 5 minutes.
  11. Centrifuge at 13000 rpm for 10 minutes.
  12. Pour off supernatant and wash pellet with 500 µL of 70% ethanol, ensuring to resuspend the pellet.
  13. Centrifuge at 13000 rpm for 1 minutes.
  14. Decant ethanol and place tubes upside down on paper towel to dry off excess ethanol.
  15. Dissolve pellet in 50 µl elution buffer, place tube on bench for 4 hours.
  16. Centrifuge at 13000 rpm for 2 minutes.
  17. Discard the supernatant, DNA is in the pellet.

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