Inoue comp cells preparation

From Inoue, Gene 96: 23-28 (1990).

Reagents

• Inoue Buffer: 10 mM HEPES, 250 mM KCl, 15 mM CaCl2, 55 mM MnCl2, pH 6.7
  • Mix components except MnCl2, adjust volume, and pH to 6.7.
  • Then add MnCl2, dissolve, and sterile filter.
• 2xYT+MgCl2: to 0.5 L, add 2.5 mL sterile 2 M MgCl2.

Protocol

1. Streak cells onto plate and grow overnight.
2. Inoculate 50 mL starter culture and grow overnight at 37 °C.
3. Inoculate 3x 250 mL 2xYT+MgCl2 with 1, 2, and 5 mL starter culture.
4. Grow at 18 °C until OD600 ~0.6. This will take anywhere from 1.5-2 days.
5. Pour culture into pre-chilled sterile bottle and incubate on ice for 10 min. To sterilize bottles, rinse with 70% ethanol and let dry.
6. Pellet cells at 3000 xg for 10 min at 4 °C.
7. Pour off supernatant and swirl pellet in 25 mL Inoue buffer to resuspend. Add an additional 50 mL Inoue buffer and mix gently. Be very gentle when resuspending as vigorous resuspension can kill cells and reduce the transformation efficiency.
8. Incubate on ice for 10 min.
9. Repeat step 6.
10. Resuspend pellet in 10 mL Inoue buffer.
11. Add 700 µL DMSO dropwise to cell suspension while swirling.
12. Incubate on ice for 10 min.
13. Aliquot into tubes and freeze in liquid nitrogen.