Day 1:

Plate Top 10 cells and transform overnight, using same protocol to plate from glycerol stock. Incubate for 16-20 hours at 37oC.

Day 2:

1. Pick a single bacterial colony (2-3 mm in diameter) from plated Top 10 cells and transfer the colony into 25 mL of SOB medium in a 250 mL flask. Incubate the culture for 6-8 hours at 37oC in the shaker (250-300 rpm).
2. At the end of the evening, use starter culture to inoculate three 1L flasks, each with 250 mL of SOB. The first flask receives 10 mL of starter culture, the second receives 4 mL, and the third receives 2 mL. Use a foam stopper to seal – remember to flame the top before and after opening/closing.
3. Incubate all three flasks overnight at 18-22oC with moderate shaking.

Day 3:

1. In the morning, read the OD600 of all three cultures. Continue to monitor the OD every 45 minutes. When the OD600 of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the other two cultures. If the OD of all three overshoots the target concentration, dilute back down to 0.1 and regrow until it reaches 0.55.
2. Split the 250 mL cultures among three centrifuge jars. Harvest the cells by centrifugation at 2500g (3900 rpm) for 10 minutes at 4oC.
3. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes.
4. Resuspend the cells gently in 80 mL (80 divided by 3 for each jar) of ice-cold CCMB80 buffer by swirling (do not pipet or vortex).
5. Use a serological pipet to transfer all cultures into one centrifuge jar. Harvest the cells by centrifugation at 2500g (3900 rpm) for 10 minutes at 4oC.
6. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes.
7. Resuspend the cells gently in 20 mL of ice-cold CCMB80 buffer.
8. Add 1.5 mL of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.
9. Working quickly, dispense 200 uL aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -80oC until needed.

Adapted from OpenWetWare and The Parts Registry