Creating Competent E. coli Cells

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Day 1:

  1. Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.

Day 2:

  1. From this grown plate, pick a single colony and inoculate 10ml LB (+ antibiotics if applicable) in a 50ml Erlenmeyer flask. Grown overnight at 37 degrees with shaking.
  2. Have a 2L Erlenmeyer flask with 500ml LB autoclaved and ready to go for tomorrow.
  3. Have three 250ml centrifuge tubes autoclaved and ready to go. Ask Eric where these are.
  4. Autoclave 50ml of 0.1M CaCl2 in 15% glycerol (40.7ml 0.1M CaCl2 + 9.3ml of 80% glycerol)
  5. Label and set up 100 0.6ml tubes for the eventual aliquots.


Day 3:

       Important: Treat cells gently after addition of CaCl2 — no vortexing or vigorous pipetting — as this will greatly reduce the competency.
  1. Transfer 5ml of this overnight culture into the 2L flask containing 500ml LB. Grow at 37 degrees with shaking.
  2. Immediately after transferring this culture, sterile syringe filter (0.22um filter) the 50ml 0.1M CaCl2 in glycerol. Put on ice and place at 4 degrees. Additionally, chill two 50ml plastic tubes.
  3. Grow culture until OD600 is ~0.4. This will take 2-3 hours.
  4. From now on, everything must be on ice!
  5. Roughly divide the 500ml of grown E. coli into the three 250ml centrifuge tubes. Do not be tempted to only use 2 tubes; this will result in LB spilling out into the centrifuge.
  6. Put these tubes in a styrofoam box containing ice; place in the refrigerator. These need to incubate for a total of 20 minutes.
  7. Go to Superlab and turn on the large centrifuge. Ensure that it is set to 4 degrees and that it is actively decreasing in temperature.
  8. Also, turn on the tabletop swinging bucket centrifuge in Superlab. Ensure that it is set to 4 degrees and that it is actively decreasing in temperature.
  9. After the 20 minute incubation at 4 degrees, weigh out the three centrifuge tubes and balance them to the nearest 0.5g. This will take some time; keep the E. coli on ice.
  10. Centrifuge the cultures at 3000 x g at 4 degrees for 10 minutes.
  11. Turn off centrifuge. Take cultures back to the MEE lab and slowly pour off supernatant into a large graduated beaker.
  12. Resuspend the cells gently in 30ml (total) ice-cold 0.1M CaCl2 + glycerol. Transfer to the two pre-chilled 50ml tubes.
  13. Incubate cells on ice for 30 minutes at 4 degrees.
  14. After the 30 minute incubation at 4 degrees, weigh out the two 50ml tubes and balance them to the nearest 0.5g.
  15. Centrifuge for 5 minutes at 3000 x g at 4 degrees using the swinging bucket centrifuge in Superlab.
  16. Turn off centrifuge. Take cultures back to the MEE lab and slowly pour off supernatant into same large graduated beaker as before. When you have time, add enough Virkon to this to kill off any cells and dispose of.
  17. Resuspend cells gently with 8ml total ice-cold 0.1M CaCl2 in glycerol. Do not vortex!
  18. Transfer 260ul aliquots into 0.6ml tubes; flash-freeze in liquid nitrogen and store at -80 degrees. Use the electronic pipettor for doing this faster!