10B Competent Cell Preparation

Adopted from Stone Lab protocol by Christian (May 2016)

Strain = NB570, 10-Beta Cells (Streptomycin resistant)

Solutions
LB: SOB (Maniatis): RF1: RF2:
 * 1% bactotryptone
 * 0.5% yeast extract
 * 1% NaCl. Autoclave 40 min.
 * 2% bactotryptone
 * 0.5% yeast extract
 * 0.05% NaCl
 * 2.5mM KCl. pH to 7 with NaOH
 * Autoclave 40 min, then add MgCl2 and MgSO4 to 10mM before using
 * 100mM RbCl
 * 50mM MnCl2-H2O
 * 30mM KOAc
 * 10mM CaCl2-2H2O
 * 15% glycerol
 * pH to 5.8 with acetic acid (CH3COOH) and sterile filter.
 * 10mM MOPS
 * 10mM RbCl
 * 75mM CaCl2-2H2O
 * 15% glycerol
 * pH to 6.8 with NaOH and sterile filter.

Protocol
Notes: 1. Grow 5mL culture overnight in LB (from a single colony).
 * Always keep cells on ice.
 * Use Avanti J-26 XP centrifuge (Sin 112) at 4°C with JA-20 rotor.
 * I usually double this protocol to avoid making cells constantly.

2. Add 2mL overnight culture in 100mL SOB + 10mM MgCl2 + 10mM MgSO4. Note: I’ve substituted SOC or 2XTY supplemented with MgCl2/MgSO4 instead of the SOB without any issues.

3. Grow ~2 hours @ 37C.

4. Check OD. When OD is between 0.4-0.6, cells are ready to harvest.

5. Split into 2x 50mL tubes. a. Note: use JA-20 rotor tubes.

6. Ice cells 10 minutes.

7. Spin 3K for 10 minutes @ 4C. Remove sup.

8. Resuspend each pellet with 16mL ice cold RF1 (16mL per 50mL tube). Note: Can vortex gently or use pipetteman. Be gentle.

9. Ice cells 15 minutes.

10. Spin 3K for 10 minutes @ 4C. Remove sup.

11. Resuspend each pellet with 4mL ice cold RF2 (4mL per 50mL tube).

12. Ice cells 15 minutes.

13. Make 100uL aliquots on ice (or dry ice) in 500uL microcentrifuge tubes. a. Note: I use metal racks to chill lots of tubes at once.

14. Flash freeze aliquots in liquid N2.

15. Store @ -80C. 16. Test competency. Transforming 100ng of plasmid should give lawn.