Day 1

1. Place the following items at 4°C overnight:

   • >50 mL "rinse buffer" (100 mM CaCl₂)
   • >8 mL "storage buffer" (60 mM CaCl₂, 10 mM Tris, 15% glycerol, pH 7.5)
   • 1 50 mL pipet
   • 1 10 mL pipet
   • >80 PCR tubes
   • tips for aliquoting (repeater is easier)
   • tube rack

2. Pick a colony and grow overnight in 4 mL LB at 37°C with shaking.

   If you want to do the prep in one day, you can pick a colony and grow it in 2 mL LB until it reaches log phase (probably 5-6 h, but variable) instead of doing an overnight culture.

Day 2

1. Inoculate 100 mL LB with 1 mL overnight culture.

2. Grow the cells at 37°C with shaking at 225 rpm to an OD600 of 0.375 (~1h30).

3. Spin the cells at 3500g for 10 min at 4°C.

4. Gently resuspend the pellet in 50 mL rinse buffer.

5. Rest the cells on ice for 1h.

6. Spin the cells at 3500g for 10 min at 4°C.

7. Gently resuspend the pellet in 8 mL storage buffer.

8. Rest the cells on ice for 30 min.

9. Aliquot the cells in ~80 100 μL portions.

10. Flash-freeze in N₂(ℓ) and store at -80°C.