IGEM:The Citadel/Notebook/Notes Brian/06-03-10

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  • Completed a consensus protocol from the Electrocompetent Cell Preperation on 05-31-10

Contents

Protocol for Electrocompetent Cells

This protocol is adapted from the Knight Lab procedure by Austin Che and is intended for use in the Genetics and Microbiology labs at The Citadel. It prepares enough electrocompetent TOP10 E.coli for a small lab to conduct transformations for the entirety of an iGEM summer project.

Materials

  • -80°C freezer
  • 4°C refrigerator
  • 37°C incubator
  • Spectrophotometer
  • Refrigerated centrifuge with...
    • a rotor that accepts 250mL culture tubes in a swinging bucket configuration
    • a rotor that accepts 50mL culture tubes in stationary configuration
  • 1L LB Lennox (supplementation with an antibiotic is optional)
  • 1L sterile deionized water chilled to 4°C
  • 75mL sterile 10% glycerol in deionized water chilled to 4°C
  • 4 ice buckets with ice
  • 5 lbs dry ice
  • 500mL 90% ethanol
  • Approximately 100 500μL plastic tubes chilled to -80°C
  • 50mL culture tube for starter culture
  • 2L flask for culture
  • 250mL polypropylene tubes for centrifugation chilled to 4°C
  • Eight 50mL conical plastic tubes for centifugation chilled to 4°C
  • Pipets chilled to 4°C
  • Three 10mL pipet bulbs chilled to 4°C
  • 2 or more 2mL cuvettes
  • Microcentrifuge tube tray prechilled to -80°C


Procedure

  1. Prechill both rotors, the ddH2O, the 10% glycerol, the pipette bulbs, and the pipette tips to 4°C. Prechill the tube tray to -80°C.
    Autoclave all flasks and any tubes that require it. Prechill the 250mL and 50mL tubes to 4°C.
  2. Inoculate 10mL LB Lennox medium and grow for 16-17 hours at 37°C at 230 RPM.
  3. Add the 10mL overnight culture to the 1L flask of LB Lennox medium and incubate at 37°C at 230 RPM until the 600nm OD is between 0.5 and 0.75. This should occur between 3 and 4 hours after inoculation.
    For OD measurements, use sterile 2mL cuvettes.
    Zero the spectrophotometer to a sample from the original LB Lennox medium before measuring.
    Take the first OD measurement 2 hours after inoculation. Take another measurement 30 minutes later to gauge the growth factor and plan future measurements.
  4. Pour the culture into 4 250mL centrifuge tubes.
  5. Place the tubes on ice for between 15 and 60 minutes. Longer incubation times may lead to higher competency.
    For the following steps, it is important to keep cells cold at all times. Have lots of ice on hand and always places the tubes in the buckets when they are not in the chilled centrifuge. Be sure to remove all the supernatant in each step to eliminate residual ions.
  6. Using the swinging bucket rotor, centrifuge the tubes for 10 minutes at 2000G at 4°C (We set our machine to 3300 RPM. See additional notes for the specs.)
  7. Remove supernatant and gently resuspend pellets with 100mL cold ddH2O per tube.
    Initially add 10-20mL of water and resuspend by pipetting. Then add the rest of the water.
  8. Centrifuge for 15 minutes at 2000G at 4°C (We set our machine to 3300 RPM.)
  9. Remove supernatant and gently resuspend pellets with 100mL cold ddH2O per tube.
    Initially add 10-20mL of water and resuspend by pipetting. Then add the rest of the water.
  10. Divide culture solution into eight 50mL conical tubes.
  11. Place on ice for 30 minutes.
  12. Replace swinging bucket rotor for 250mL tubes with a stationary rotor that fits the 50mL tubes.
  13. Centrifuge for 15 minutes at 2000G at 4°C (We set our machine to 4300 RMP.)
  14. Remove supernatant and gently resuspend pellets with 6.25mL cold 10% glycerol per tube.
  15. Divide culture solution into four 50mL conical tubes.
  16. Place on ice for 30 minutes.
  17. Centrifuge for 15 minutes at 1500G at 4°C (We set our machine to 3700 RMP.)
  18. Remove supernatant and gently resuspend pellets with 500μl cold 10% glycerol per tube.
  19. Resuspend the cells in a final volume of approximately 2ml in a 50ml conical-bottom tube.
  20. Aliquot 25μL each into 500 μL microcentrifuge tubes. Make sure to keep those tubes on ice as you are performing the transfer.
  21. Shock freeze each cell suspension tube for 30 to 60 seconds in a bath of dry ice and ethanol.
  22. Store the tubes at -80°C. Keep tubes in the prechilled microcentrifuge tube tray.

Additional Notes

  • Label every container all of the time, especially centrifuge tubes. This will help with balancing the centrifuge and maintaining overall zen.
  • Use the conical-bottomed centrifuge tubes over the flat-bottomed ones, if available. This will result in better pellet formation. If you are using flat-bottom tubes, ensure that they are polypropylene and not the plastic ones, which are prone to breaking. (Shetty, R.)
  • Electocompentent cells can be stored for long periods of time in the -80°C freezer. However, do not remove cells from freezer and then refreeze for future use. Doing so can reduce competency by 30%. (Knight, T.)
  • In The Citadel Genetics and Microbiology labs, we use the Marathon 21000R refrigerated centrifuge by Fischer Scientific®. We utilize two rotors: a swinging bucket rotor (#04-976-006) that accepts our Nalegne® flat-bottomed 250mL polypropylene tubes, and a stationary rotor (#04-976-011) that accepts our Fischer® conical 50mL polypropylene tubes. The spin speeds we have listed in our protocol are suitable for our rotors only. If you are using a different centrifuge or rotors, consult your user's manual before use.
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