Competent Cells

Making Competent Cells

Materials

• Desired cells
• 1-2L ice cold autoclaved water
• 1L 2YT media
• 2x Erlenmeyer flasks, autoclaved
• Sterile centrifuge tubes large enough to hold 0.2-1L of culture

Procedure

NOTE: The cells must be kept as close to 0°C as possible after they are chilled. All utensils must be completely sterile, as no antibiotics are added to the competent cells, and contamination is a serious danger.

1. Incubate two 5mL overnight cultures of the desired cells in 2YT media at 37°C.
2. Use the cultures to inoculate two 100-500mL cultures of 2YT in autoclaved Erlenmeyer flasks. Incubate for 2-4 hours.
3. Transfer to centrifuge tubes (balanced), chill on ice for 20-30 minutes, and centrifuge at 4000xG for 10 minutes.
4. Decant supernatant. Resuspend pellet in an equal volume of ice-cold autoclaved water. Centrifuge again.
5. Decant, resuspend pellet in a half-volume of ice-cold water. Centrifuge.
6. Decant, resuspend pellet in 1-5mL ice-cold 10% glycerol. Centrifuge.
7. Decant, resuspend in 0.5-1mL 10% glycerol.
8. Flash-freeze 50μL aliquots in dry ice/ethanol and store at -80°C.
9. To be sure that the process was successful, perform positive and negative transformation controls using your new cells.