Difference between revisions of "Team:Carnegie Mellon/Protocols"

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   </ol></div>
 
   </ol></div>
  
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<div id = "estrogen_sensor">Estrogen Sensor</div>
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<div id = "flip_estrogen_sensor">Estrogen Sensor</div>
 
<div id = "estrogen_sensor_p">Estrogen Sensor Protocol</div>
 
<div id = "estrogen_sensor_p">Estrogen Sensor Protocol</div>
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<!-- ________________________________ Miniprep Protocol __________________________ -->
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<div id = "flip_miniprep">MiniPrep Protocol</div>
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<div id = "miniprep_p">
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<div><b>Minipreps of MACH Cells Expressing Flourescence</b></div>
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<div><b><u>Purpose:</u></b> To isolate plasmid DNA from MACH cells.</div>
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<p><b><u>Procedure:</u></b></p>
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<ol>
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<li>Set up overnight cultures for miniprep.</li>
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<li>Make the following reaction recipe:
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<ul>
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<li>5 mL LB</li>
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<li> 5 µL Chlorophenical</li>
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<li>1 µL overnight colony</li>
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</ul></li>
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<li>Incubate in 37°C for 16-18 hours.</li>
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<li>Follow the <a href = "https://tools.lifetechnologies.com/content/sfs/manuals/MAN0012655_GeneJET_Plasmid_Miniprep_UG.pdf">Life Technologies Miniprep Kit Protocol</a>.</li>
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</ol>
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</div>
 
</body>
 
</body>
  
 
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</html>

Revision as of 17:16, 6 August 2015

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His-Tag Soluble Protein Extraction
  1. Centrifuge 1.5 ml of culture for 1min full speed at 15000 rpm.
  2. Remove supernatant.
  3. Add 250 µl of extraction buffer (1% octyl-beta-thioglucoside in 10mM Tris-Cl, pH7.5).
  4. Incubate at room temperature for 10 min.
Wash Beads
  1. Remove 50 µl of Ni-NTA in 10 mM Trischloride bead solution and place in new tube for each purification.
  2. Add 1 mL buffer (see table).
  3. Centrifuge at 800 rpm for 30 seconds.
  4. Remove supernatant.
  5. Repeat 3 times and resuspend in 1 mL of Bead Wash Solution.
Estrogen Sensor
Estrogen Sensor Protocol
MiniPrep Protocol
Minipreps of MACH Cells Expressing Flourescence
Purpose: To isolate plasmid DNA from MACH cells.

Procedure:

  1. Set up overnight cultures for miniprep.
  2. Make the following reaction recipe:
    • 5 mL LB
    • 5 µL Chlorophenical
    • 1 µL overnight colony
  3. Incubate in 37°C for 16-18 hours.
  4. Follow the Life Technologies Miniprep Kit Protocol.