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

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<!-- __________________________ Restriction Enzyme Digest Protocol _____________ -->
 
<!-- __________________________ Restriction Enzyme Digest Protocol _____________ -->
 
<div id = "flip_restriction_enzyme_digest">Restriction Enzyme Digest Protocol</div>
 
<div id = "flip_restriction_enzyme_digest">Restriction Enzyme Digest Protocol</div>
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<div id = "restriction_enzyme_digest_p">
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<div class = "title">Restriction Enzyme Digest</div>
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<div class = "procedure">Procedure:</div>
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<ol>
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<li>Prepare the following restriction enzyme digestion solution for J23108, J23109, J23111.</li>
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<table>
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<tr>
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<td><b><font color = "orange">Reagent</font></b></td>
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<td><b><font color = "orange">Amount (µL)</font></b></td>
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</tr>
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<tr>
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<td>10X Fast Digest Buffer</td>
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<td>2</td>
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</tr>
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<tr>
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<td>Plasmid DNA</td>
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<td>12</td>
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</tr>
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<tr>
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<td>Restriction Enzyme XbaI</td>
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<td>1</td>
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</tr>
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<tr>
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<td>Restriction Enzyme SpeI</td>
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<td>1</td>
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</tr>
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<tr>
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<td>Water (to bring up to volume)</td>
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<td>2</td>
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</tr>
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<td><b>Total Volume</b></td>
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<td><b>18</b></td>
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</tr>
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</table>
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<li>Digest at 37 °C for 1 hour.</li>
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<li>Follow the <a href = "https://www.lifetechnologies.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/fastdigest-thermo-scientific.html">Life Technologies Restriction Enzyme Digest Protocol</a>.</li>
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Revision as of 16:15, 10 August 2015

Under Construction.

This is almost as well-documented as Kim Kardashian's wedding.

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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.
Restriction Enzyme Digest Protocol
Restriction Enzyme Digest
Procedure:
  1. Prepare the following restriction enzyme digestion solution for J23108, J23109, J23111.
    2. Reagent Amount (µL)
    10X Fast Digest Buffer 2
    Plasmid DNA 12
    Restriction Enzyme XbaI 1
    Restriction Enzyme SpeI 1
    Water (to bring up to volume) 2
    Total Volume 18
  2. Digest at 37 °C for 1 hour.
  3. Follow the Life Technologies Restriction Enzyme Digest Protocol.