Team:Calgary/Notebook/ProtocolManual/DNA

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<h1>DNA Protocols</h1>
<h1>DNA Protocols</h1>
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<b>Preparing Chemically Competent <i>E. coli</i> Cells</b>
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<li>Inoculate 5-10mL LB with Top10 E. coli culture at 37oC shaking over night</li>
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<li>Subculture 1mL of bacteria into 50mL LB at 37oC shaking until OD<sub>600</sub> is 0.4-0.6 (~2.5 hr)</li>
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<li>Centrifuge the subculture at max for 20 min at 4oC</li>
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<li>Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)</li>
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<b>Rehydrating Registry DNA</b>
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<li>Add 10μL of ddH2O to appropriate well [will turn orange]</li>
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<li>Incubate at RT for 10 min</li>
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<li>Use 1μL of DNA to transform cells</li>
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</ul>
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Store plates in -20oC
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Revision as of 17:49, 10 October 2014

DNA Protocols

Preparing Chemically Competent E. coli Cells

  1. Inoculate 5-10mL LB with Top10 E. coli culture at 37oC shaking over night
  2. Subculture 1mL of bacteria into 50mL LB at 37oC shaking until OD600 is 0.4-0.6 (~2.5 hr)
  3. Centrifuge the subculture at max for 20 min at 4oC
  4. Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)

    5. Rehydrating Registry DNA

    1. Add 10μL of ddH2O to appropriate well [will turn orange]
    2. Incubate at RT for 10 min
    3. Use 1μL of DNA to transform cells
      4. Store plates in -20oC

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