Difference between revisions of "Team:Austin UTexas/Protocols"

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<h2>Accordion</h2>
 
<h2>Accordion</h2>
  
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<button class="accordion">Section 1</button>
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<button class="accordion">Electroporation of <i>L. plantarum</li></button>
 
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   <h4>Materials Needed</h4>
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  </br>
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  <ul>
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  <li>MRS liquid media</li>
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  <li>L. plantarum cells</li>
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  <li>Glass culture tube</li>
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  <li>CO2 incubator</li>
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  <li>MRS liquid media, containing 0.5 M sucrose</li>
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  <li>Spectrophotometer</li>
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  <li>Centrifuge</li>
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  <li>Vortex</li>
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  <li>50 mL Falcon conical tubes (2)</li>
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  <li>30% PEG 8000</li>
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  </ul>
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  </br>
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  <h4>Preparation of Recipient Cell Stocks</h4>
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  <ol>
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  <li>Inoculate 10-25 mL of -80 &#2103 L. Plantarum stocks in MRS broth at 37 &#2103 CO2 incubator, overnight without shaking.</li>
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  <li>Subculture by incubating culture in a 200 mL of prewarmed MRS broth. Add culture until OD600 is 0.1.</li>
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  <li>Subcultures should be grown to an OD600 of 0.6 (4-8 hours).</li>
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  <li>For glycine supplementation, the MRS broth should contain 0.5%-1.0% glycine.</li>
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  <li>For NaCl supplementation, inoculate subcultures with 0.9M of NaCl.</li>
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  <li>Harvest cells from the subculture by centrifugation at 4 &#2103 for 10 min. at 7000 rpm after inoculation.</li>
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  <li>Rinse the cells in 200 mL of sterile, distilled water (~4 &#2103) and centrifuge again.</li>
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  <li>Resuspend the pellet in 2-3 mL of cold, sterile, distilled water and aliquot 1 mL volumes to 1.5 mL microcentrifuge tubes.</li>
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  <li>Centrifuge aliquots at 15,000 rpm for ~90 seconds and remove the supernatant.</li>
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  <li>Rinse the cells twice with 1 mL of cold, distilled, sterile water. Then rinse with 1 mL of cold, sterile 30% PEG-8000 solution. Remove the supernatant.</li>
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  <li>Suspend cells in 0.5-0.6 mL of 30% PEG for storage at -80 &#2103. These should retain viability for up to 2 years.</li>
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  </ol>
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</div>
 
</div>
  

Revision as of 20:25, 19 October 2017

Accordion

Materials Needed


  • MRS liquid media
  • L. plantarum cells
  • Glass culture tube
  • CO2 incubator
  • MRS liquid media, containing 0.5 M sucrose
  • Spectrophotometer
  • Centrifuge
  • Vortex
  • 50 mL Falcon conical tubes (2)
  • 30% PEG 8000

Preparation of Recipient Cell Stocks

  1. Inoculate 10-25 mL of -80 &#2103 L. Plantarum stocks in MRS broth at 37 &#2103 CO2 incubator, overnight without shaking.
  2. Subculture by incubating culture in a 200 mL of prewarmed MRS broth. Add culture until OD600 is 0.1.
  3. Subcultures should be grown to an OD600 of 0.6 (4-8 hours).
  4. For glycine supplementation, the MRS broth should contain 0.5%-1.0% glycine.
  5. For NaCl supplementation, inoculate subcultures with 0.9M of NaCl.
  6. Harvest cells from the subculture by centrifugation at 4 &#2103 for 10 min. at 7000 rpm after inoculation.
  7. Rinse the cells in 200 mL of sterile, distilled water (~4 &#2103) and centrifuge again.
  8. Resuspend the pellet in 2-3 mL of cold, sterile, distilled water and aliquot 1 mL volumes to 1.5 mL microcentrifuge tubes.
  9. Centrifuge aliquots at 15,000 rpm for ~90 seconds and remove the supernatant.
  10. Rinse the cells twice with 1 mL of cold, distilled, sterile water. Then rinse with 1 mL of cold, sterile 30% PEG-8000 solution. Remove the supernatant.
  11. Suspend cells in 0.5-0.6 mL of 30% PEG for storage at -80 &#2103. These should retain viability for up to 2 years.

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Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.