Team:Grinnell/Notebook/Protocols

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===Competent Cells===<html><a name="Competent"></a>
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<html><h3><a name="Competent">Competent Cells</a></h3>
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<li>Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).</li>
<li>Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).</li>
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<li>Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80 degrees C.</li>
<li>Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80 degrees C.</li>
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<h3>Plasmid
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<h3><a name="Transformation">Plasmid Transformation</a></h3>
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<ol>
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<li>Thaw 100uL aliquots of competent cells on ice.</li>
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<li>Add 10uL DNA to cells.</li>
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<li>Incubate tubes on ice for 30min.</li>
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<li>Incubate tubes at 42 degrees C for 90sec.</li>
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<li>Incubate tubes on ice for 2min.</li>
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<li>Add 300mL LB to cells and incubate shaking at 37 degrees C for 1hr.</li>
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<li>Spread cells on selective media</li>
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<li>Incubate plates overnight at 37 degrees C.</li>
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</ol>
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Revision as of 21:50, 8 June 2011

Competent Cells

  1. Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).
  2. Chill cells on ice for 15-120min.
  3. Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4 degrees C. Discard supernatant.
  4. Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
  5. Harvest cells by cetrifugation. Discard supernatant.
  6. Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80 degrees C.

Plasmid Transformation

  1. Thaw 100uL aliquots of competent cells on ice.
  2. Add 10uL DNA to cells.
  3. Incubate tubes on ice for 30min.
  4. Incubate tubes at 42 degrees C for 90sec.
  5. Incubate tubes on ice for 2min.
  6. Add 300mL LB to cells and incubate shaking at 37 degrees C for 1hr.
  7. Spread cells on selective media
  8. Incubate plates overnight at 37 degrees C.

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