Difference: ProtocolsElectrocompetentCells (16 vs. 17)

Revision 172018-12-10 - KateElston

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META TOPICPARENT name="ProtocolList"

Electrocompetent _E. coli

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  1. Grow an overnight culture of each strain in LB medium.
  2. Prepare 100 ml of fresh LB medium in a 500 ml flask for each strain.
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  1. Inoculate with 1 mL of the overnight, stationary-phase culture.
    • In general, inoculate to OD600 of ~0.05.
  2. Grow the cells for approximately 3-4 hours, until they reach mid-exponential phase.
    • In general, this is an OD600 of ~0.6.
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  1. Inoculate with overnight, stationary-phase culture to an OD of ~0.05.
  2. Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.
    • In general, this is an OD600 of 0.4-0.6.
  3. ~30 min before you plan to prepare your cells, set the centrifuge to 4C.
 
  1. Transfer the cells to 2x 50 ml Falcon conical tubes.
  2. Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
  3. Wash by adding 40 ml of chilled 10% glycerol to each tube, then vortexing vigorously to resuspend the pellet. Centrifuge for 3.5 minutes. Remove promptly and pour off supernatant. Repeat for at least four wash cycles in 10% glycerol.
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  1. To the electrocompetent cells, add 1-3 μl of DNA (<100 ng of DNA).
  2. Mix by gently flicking the tube containing the electrocompetent cell + DNA mixture. Let the mixture sit on ice for 1-10 minutes.
  3. Pipette the mixture into a chilled cuvette, making sure that the mixture is at the bottom of the cuvette by gently tapping the cuvette on a flat surface.
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    • Be sure to wipe any condensation off the sides of the cuvette before electroporation.
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    • Be sure to wipe any condensation off the sides of the cuvette before electroporation.*
 
  1. Place the cuvette in the pulser and press the "Pulse" button.
    • For the TOP10 Electrocomp™ E. coli cells, the Ec l setting is fine.
  2. After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
 
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