Team:Paris Bettencourt/Protocols

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<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
<li>Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
<li>Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
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Note: Frozen cells are only good once.Do not refreeze cells once thawed.
Note: Frozen cells are only good once.Do not refreeze cells once thawed.
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== <b> Protocol 3:</b> Glycerol Stocks ==
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<li>Pick Single colonies from agar plates
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<li>Innoculate 5ml LB broth overnight.
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<li>Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.
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<li>Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.
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Revision as of 15:11, 22 July 2013

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Protocols

Protocol 1: Heat Shock Transformation

  1. Thaw 20 ul of Chemically Competent Cells on Ice
  2. Add 2 ul DNA (intact plasmid)
  3. Incubate on Ice for 30 minutes
  4. Incubate at 42C for 45 seconds
  5. Incubate on Ice for 2 minutes
  6. Add 200 ul of LB broth
  7. Incubate at 37C for 1 hour in shaker
  8. Plate on Agar supplemented with appropriate antibiotics.

Protocol 2: CaCl2 Competent Cells

This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.

Note: Never vortex competent cells.

Resuspend by pipetting with large Pasteur pipettes.

    The night before:

  1. The night before, inoculate a 5 ml culture and grow overnight with selection.

    The day of:

  2. Dilute cells ~ 1:200 into selective media.
    For this example add 250 ul to 50 ml of selective media.
    Note: The protocol is easily scaled to increase the number of cells.
  3. Grow the cells to an OD600 of 0.6 – 0.7.
    Use a large flask, 500ml, for good aeration.
    Use a baffled flask for fastest growth.
    This takes about 3 hours depending on the cells.
    Medium-heavy cloudiness by eye is fine.
  4. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
  5. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
  6. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
  7. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
  8. Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
Note: Frozen cells are only good once.Do not refreeze cells once thawed.

Protocol 3: Glycerol Stocks

  1. Pick Single colonies from agar plates
  2. Innoculate 5ml LB broth overnight.
  3. Add 750ml of overnight culture to 250ml of 60% glycerol in a cryotube.
  4. Make two sets of Glycerol stocks freeze one at -20ºC and the other at -80ºC.
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