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. 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 250 mL culture tubes in a swinging bucket configuration
  • a rotor that accepts 50mL culture tubes in stationary configuration
• 500 mL LB Lennox supplemented with the antibiotic which the Biobrick part will confer resistence for
• 750 mL sterile deionized water chilled to 4°C
• 75 mL sterile 10% glycerol in deionized water chilled to 4°C
• 4 ice buckets with ice
• 5 lbs dry ice
• 500 mL 90% ethanol
• Approximately 100 500 uL plastic tubes chilled to -80 °C
• 50 mL culture tube for starter culture
• 2 L flask for culture
• 250 mL polypropylene tubes for centrifugation chilled to 4°C
• Eight 50 mL conical plastic tubes for centifugation chilled to 4°C
• Pipets chilled to 4°C
• Three 10 mL pipet bulbs chilled to 4°C
• 2 mL 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 250 mL and 50 mL tubes to 4°C.
2. Inoculate 10 mL LB Lennox medium and grow for 16-17 hours at 37°C with 230 RPM rotation.
3. Add the 10 mL overnight culture to a 1 L flask of LB Lennox medium and incubate at 37°C with 230 RPM roatation 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 2 mL 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 mearurement 30 minutes later to gauge the growth factor and plan future measurements.
4. Pour the culture into four 250 mL 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 centrifuge. Also be sure to remove all the supernatant in each step to eliminate residual ions.
   
6. Using the swinging bucket rotor, centrifuge tubes for 10 mins at 2000 g at 4°C (We set our machine to 3300 RPMs.)
7. Remove supernatant and gently resuspend pellets with 100 mL cold ddH₂O per tube.
   Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
8. Centrifuge for 15 mins at 2000 G at 4°C (We set our machine to 3300 RPMs.)
9. Remove supernatant and gently resuspend pellets with 100mL cold ddH₂O per tube.
   Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
10. Divide culture solution into 8 50 mL conical-bottom tubes.
11. Hold on ice for 30 minutes.
12. Replace swinging bucket rotor for 250 mL tubes with a stationary rotor that fits the 50 mL tubes.
13. Centrifuge for 15 mins at 2000 G at 4°C (We set our machine to 4300 RMPs.)
14. Remove supernatant and gently resuspend pellets with 6.25 mL cold 10% glycerol per tube.
15. Divide culture solution into 4 50 mL conical-bottom tubes.
16. Hold on ice for 30 minutes.
17. Centrifuge for 15 mins at 1500 G at 4°C (We set our machine to 3700 RMPs.)
18. Remove supernatant and gently resuspend pellets with 500 μl cold 10% glycerol per tube.
19. Resuspend the cells in a final volume of approximately 2 ml in a 50 ml conical-bottom tube.
20. Aliquot 25 μL per tube (tubes on ice) into 500 μL microcentrifuge tubes.
21. Shock freeze each cell suspension tube for 30 to 60 seconds in a dry ice and ethanol bath.
22. Store in -80°C. Keep tubes in prechilled microcentrifuge test tube tray.

NOTES

• Label every container all of the time, especially centrifuge tubes. This will help with balancing the centrifuge and overall zen.

• Use the conical-bottomed 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 plastic ones, which are prone to breaking.

• 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%.