Team:Calgary/Notebook/Protocols/Process8

From 2011.igem.org

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TITLE=Plasmid Extraction|
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TITLE=Nuclear DNA Extraction from Algae|
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<p>
 
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This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.</p>
 
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<br>
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<h4>Total DNA Isolation Protocol</h4>
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<h4> Reagents </h4>
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<ol>
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<li>LB broth, pH 7</li>
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<li>10g tryptone</li>
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<li>5g yeast extract</li>
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<li>5g NaCl</li>
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<li>20% sucrose (autoclaved)</li>
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<li>Triton X-100</li>
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<li>500mM EDTA stock (pH 8.0)</li>
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<li>Tris-HCl 50mM</li>
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<li>NaCl 3M</li>
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<li>Isopropanol</li>
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<li>Autoclaved Milli-Q water</li>
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</ol>
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<br>
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<p><h5>Reagents and Materials</h5></p>
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<h4> Procedure </h4>
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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>
<ol>
<ol>
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<li>Inoculate a loopful of Pseudomonas sp. at 25oC, in 10mL LB broth (10g tryptone; 5g yeast extract, 5g NaCl, 1000mL distilled H2O, pH 7.0), and incubate for 16-18hr.</li>
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<li>Place 1.5 ml of algae culture into centrifuge tube.</li>
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<li>Centrifuge 1.5 ml of a 16-18hr bacterial culture for 1 min at 11,500 x g in a polypropylene centrifuge tube.</li>  
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<li>Spin down cells at 5000 rpm for 1 minute.</li>
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<li>Remove supernatant by aspirating, leaving the pellet as dry as possible.</li>
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<li>Pipette off supernatant.</li>
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<li>Add to each tube, 400µL of 8% sucrose, 5.0% Triton X-100, 50 mM EDTA, and 10mM Tris HCI (pH 8.0). Mix well.</li>
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<li>Add 400 µl extraction buffer.</li>
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<li>Add 50µL of freshly prepared lysozyme solution (10mg/mL in 10mM Tris HCl, pH8), mix by inverting 3X.  Lysozyme digests bacterial cell wall.</li>
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<li>Incubate for 15 minutes at 50ºC, inverting tubes several times during incubation.</li>
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<li>Immediately incubate at 100oC for 10, 20, 40, 80s.</li>
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<li>Centrifuge at 13000 rpm for 5 minutes.</li>
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<li>Spin for 10min 11,500Xg at room temp.</li>
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<li>Transfer supernatant to new tube.</li>
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<li>Remove pellet with sterile forceps.</li>
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<li>Add 400 µl isopropanol.</li>
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<li>Add to supernatant, 50µL of cold 3M NaAc and 420µL of cold isopropanol.</li>
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<li>Invert several times.</li>
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<li>Incubate 30min at -20oC.</li>
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<li>Place on ice for 5 minutes.</li>
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<li>Centrifuge 15min for 11,500Xg at 4oC.</li>
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<li>Centrifuge at 13000 rpm for 10 minutes.</li>
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<li>Decant supernatant, invert and drain on clean paper towel.</li>
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<li>Pour off supernatant and wash pellet with 500 µL of 70% ethanol, ensuring to resuspend the pellet.</li>
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<li>Add 15µL of cold/4oC TE buffer (0.05M Tris, 0.01M EDTA, pH8).</li>
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<li>Centrifuge at 13000 rpm for 1 minutes.</li>
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<li>Incubate for 1hr at 4oC in dark. </li>
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<li>Decant ethanol and place tubes upside down on paper towel to dry off excess ethanol.</li>
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<li>Run a small amount of this sample on gel electrophoresis on 0.7% (w/v0) agarose.  With the rest, submit to further purification.</li>
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<li>Dissolve pellet in 50 µl elution buffer, place tube on bench for 4 hours.</li>
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<li>Centrifuge at 13000 rpm for 2 minutes.</li>
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<li>Discard the supernatant, DNA is in the pellet.</li>
</ol>
</ol>
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<br>
 
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<h4>Further purification (Plasmid from putida)</h4>
 
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<ol>
 
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<li>Layer the resuspended DNA on a 2.5mL bed of saturated CsCl in a polymer tube.  Centrifuge for 14hr at 14 000 rpm in a fixed-angle 30 rotor at 2°C.  After the run, ~25mL of liquid can be discarded from the top without disturbing the remainder.  Mix the lower part to form the concentrated lysate.</li>
 
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<li>Slowly dissolve ~5.7g CsCl to the concentrated lysate, until the refractive index is 1.399.  Mix solution with Syber-safe or gel-red.  Centrifuge for 40hr in a Spinco fixed-angle rotor 50 at 105 000 x g at 12°C.  After this run, 2 well-separated bands should be able to be seen.  Alternatively, do only this spin where DNA mixed with CsCl is concentrated to a refractive index of 1.399 with the fluorescent DNA stain.</li>
 
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<li>Because the DNA was infused with dye, 2 well-separated bands will appear.  The upper band is linear and non-circular DNA (junk).  The lower band is the plasmid of interest.  Remove the upper layer with a micropipette.  After it is removed, pool bands from several tubes centrifuge again SW50.1 rotor for 20h at 40 000rpm.  Again there will be 2 bands and the lower band is the desired intact plasmid.</li>
 
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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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