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

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

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

Materials

• -80°C freezer
• 37°C incubator
• Refrigerated centrifuge with...
  • a rotor that accepts 250 mL culture tubes in a swinging bucket configuration
  • a rotor that accepts 50mL culture tubes
• 500 mL LB Lennox supplemented with antibiotic which the Biobrick part will confer resistence for
• 750 mL sterile deionized water chilled to 4°C
• 50 mL sterile 10% glycerol in deionized water chilled to 4°C
• 4 ice buckets with ice
• 5 lbs dry ice
• 500mL 90% ethanol
• Many 1.5 mL plastic tubes chilled to -80 °C
• 14 mL culture tube for starter culture
• 2 1L flasks for culture
• 250 mL polypropelyne tubes for centrifugation chilled to 4°C
• Pipets chilled to 4°C
• Many 0.5 mL tubes chilled to 4°C

Procedure

1. Prechill all tubes and pipets at 4°C or -80°C as appropriate.
   Autoclave all flasks.
2. Inoculate 5mL LB Lennox medium and grow for 16 hours at 37°C with 240 RPM rotation.
3. 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.
4. Fast cool the centrifuge with the correct rotor to 4°C
5. Pour the culture into two 250 mL centrifuge tubes.
6. 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.
   
7. Centrifuge for 10 mins at 2000g at 4°C (That's 3282.4 RPMs on our machine.)
8. 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.
9. Centrifuge for 15 mins at 2000g at 4°C (That's 3282.4 RPMs on our machine.)
10. 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.
11. Hold on ice for 30 minutes
12. Centrifuge for 15 mins at 2000g at 4°C (That's 3282.4 RPMs on our machine.)
13. Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol.
    This can be optionally transferred to a 50 mL conical tube.
14. Hold on ice for 30 minutes
15. Centrifuge for 15 mins at 1500g at 4°C (That's 2844 RPMs on our machine.)
16. Remove the supernatant and add 500 μl of 10% glycerol
17. Resuspend the cells in a final volume of approximately 1 ml
18. Aliquot 50 μL per tube (tubes on ice)
19. Shock freeze cell suspensions in a dry ice and ethanol bath.
    One website recommended against using liquid nitrogen but did not justify this recommendation.
20. Store at -80°C

NOTE: Use the conical-bottomed tubes over the flat-bottomed ones for the swinging buckets in the centrifuge to prevent the tubes from breaking.