Creating Competent E. coli Cells

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.

Day 3:

1. Transfer 5ml of this overnight culture into the 2L flask containing 500ml LB. Grow at 37 degrees with shaking until OD600 is ~0.4. This will take 2-3 hours.
2. From now on, everything must be on ice!
3. 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.
4. Put these tubes in a styrofoam box containing ice; place in the refrigerator. These need to incubate for a total of 20 minutes.
5. 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.
6. Also, turn on the tabletop bucket centrifuge in Superlab. Ensure that it is set to 4 degrees and that it is actively decreasing in temperature.
7. 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.
8. Centrifuge the cultures at 3000 x g at 4 degrees for 10 minutes.
9. Take cultures back to the MEE lab and