IGEM:The Citadel/Notebook/Notes Brian/05-31-10

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May 31

This is a continuation of our work from 05-30-10. Text with strikethrough was accomplished on a prior date. All other text was performed on 05-31-10.

Protocol for Electrocompetent Cells

Procedure

  1. Autoclaved all flasks and tubes.
    The 250mL tubes were autoclaved with caps off, so to ensure sterility we've stored them on ethanol-soaked paper.
  2. Prechilled the rotor at 4°C.
    All tubes and pipets will be chilled tomorrow morning upon arrival.
  3. Inoculated 5mL LB Miller medium and placed in incubator for overnight growth at 37°C with 240 RPM.
    Austin Che calls for LB Lennox to lessen salt content. We had only LB Miller at the time of inoculation and have attempted to use that; we'll post an update! For tomorrow's 450mL culture, we've prepared 500mL of LB Lennox.
  4. Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours.
    For recA- strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.
  5. Fast cool the centrifuge with the correct rotor to 4°C
  6. Pour the culture into two 225 mL centrifuge tubes. (Reshma Shetty advises conical-bottomed tubes over flat-bottomed ones for the centifuge's swinging buckets to prevent the tubes from breaking. We tested flat-bottomed polypropylene tubes made by Nalgene and they did not break under the conditions specified by this protocol.)
  7. Place the tubes on ice for 15 minutes.
    This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.
    For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.
  8. Centrifuge for 10 mins at 2000g at 4°C
  9. Remove supernatant and gently resuspend pellets with 200mL cold sterile water.
    Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
  10. Centrifuge for 15 mins at 2000g at 4°C
  11. Remove supernatant and gently resuspend pellets with 200mL cold sterile water.
    Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
  12. Hold on ice for 30 minutes
  13. Centrifuge for 15 mins at 2000g at 4°C
  14. Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol.
    This can be optionally transferred to a 50 mL conical tube.
  15. Hold on ice for 30 minutes
  16. Centrifuge for 15 mins at 1500g at 4°C
  17. Remove the supernatant and add 500 μl of 10% glycerol
  18. Resuspend the cells in a final volume of approximately 1 ml
  19. Aliquot 50 μL per tube (tubes on ice)
  20. Shock freeze cell suspensions in a dry ice and ethanol bath.
    One website recommended against using liquid nitrogen but did not justify this recommendation.
  21. Store at -80°C
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