Team:Paris Bettencourt/Protocols

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<li><a onclick="showprot('#prot1')">Protocol 1</a></li>
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<li><a onclick="showprot('#prot1')">Heat Shock Transformation of <i>E. coli</i></a></li>
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<li><a onclick="showprot('#prot2')">Protocol 2</a></li>
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<li><a onclick="showprot('#prot2')">CaCl2 Competent Cells</a></li>
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<li><a onclick="showprot('#prot3')">Protocol 3</a></li>
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<li><a onclick="showprot('#prot3')">Electroporation</a></li>
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                                <li><a onclick="showprot('#prot4')"> Miniprep </a></li>
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                                <li><a onclick="showprot('#prot5')">PCR Purification</a></li>
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                                <li><a onclick="showprot('#prot6')">Gel Purification</a></li>
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                                <li><a onclick="showprot('#prot7')">Glycerol Stocks</a></li>
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                                <li><a onclick="showprot('#prot8')">Colony PCR</a></li>
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<h3> Heat Shock Transformation of <i>E. coli</i></h3>
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      <p>This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.</p>
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      <h5>Note: Never vortex competent cells. Mix cells by gentle shaking.</h5>
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      <p><ol>
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  <li>Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.</li>
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  <li>Place 20 ul of cells in a pre-chilled Eppendorf tube.
 +
    <ul>
 +
              <li><u>For an Intact Vector:</u> Add 0.5 ul or less to the chilled cells</li>
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              <li><u>For a Ligation Product:</u> Add 2-3 ul to the chilled cells.</li>
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    </ul>
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  </li>
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  <li>Mix gently by flicking the tube.</li>
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  <li>Chill on ice for 10 minutes. <em>This step is optional, but can improve yields when transforming a ligation product.</em></li>
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  <li>Heat shock at 42 &deg;C for 30 seconds.</li>
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  <li>Return to ice for 2 minutes.</li>
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  <li>Add 200 ul LB medium and recover the cells by shaking at 37 &deg;C.<br />
 +
    Another rich medium can substitute for the recovery.<br />
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    The recovery time varies with the antibiotic selection.<br />
 +
    Ampicillin: 15-30 minutes<br />
 +
    Kanamycin or Spectinomycin: 30-60 minutes<br />
 +
    Chloramphenicol: 60-120 minutes
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  </li>
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  <li>Plate out the cells on selective LB.<br />
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    Use glass beads to spread the cells.<br />
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    The volume of cells plated depends on what is being transformed.<br />
 +
    <ul>
 +
              <li><u>For an Intact Vector:</u> High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.</li>
 +
              <li><u>For a Ligation Product:</u> Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.</li>
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    </ul>
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    Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.
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  </li>
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  <li>Incubate at 37 &deg;C. Transformants should appear within 12 hrs.</li>
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      </ol></p>
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Revision as of 13:19, 19 August 2014

Paris Bettencourt 2014

Heat Shock Transformation of E. coli

This protocol can be used to transform chemically competent (i.e. from CaCl2) with a miniprepped plasmid or a ligation product.

Note: Never vortex competent cells. Mix cells by gentle shaking.

  1. Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
  2. Place 20 ul of cells in a pre-chilled Eppendorf tube.
    • For an Intact Vector: Add 0.5 ul or less to the chilled cells
    • For a Ligation Product: Add 2-3 ul to the chilled cells.
  3. Mix gently by flicking the tube.
  4. Chill on ice for 10 minutes. This step is optional, but can improve yields when transforming a ligation product.
  5. Heat shock at 42 °C for 30 seconds.
  6. Return to ice for 2 minutes.
  7. Add 200 ul LB medium and recover the cells by shaking at 37 °C.
    Another rich medium can substitute for the recovery.
    The recovery time varies with the antibiotic selection.
    Ampicillin: 15-30 minutes
    Kanamycin or Spectinomycin: 30-60 minutes
    Chloramphenicol: 60-120 minutes
  8. Plate out the cells on selective LB.
    Use glass beads to spread the cells.
    The volume of cells plated depends on what is being transformed.
    • For an Intact Vector: High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.
    • For a Ligation Product: Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.
    Note: 200 ul is the maximum volume of liquid that an LB plate can absorb.
  9. Incubate at 37 °C. Transformants should appear within 12 hrs.

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