while shaking until the optical density at 600 nm (OD 600 )
reaches 0.4–0.5 (see Note 12).
3. When cells reach the desired OD 600 , immediately place the
200 mL flask on ice, and incubate for 20–30 min (see Note
13). In preparation for the next step, place two 2 mL Eppendorf tubes and eight 15 mL centrifuge tubes on ice, and
precool the centrifuge to 4
C.
4. Transfer the culture to the eight 15 mL ice-cold centrifuge
tubes (12.5 mL in each tube). Centrifuge the bacteria at
3000 rpm (2250 Â g) for 20 min at 4
C and pour off the
supernatant (see Note 14).
5. Gently resuspend the pellet in each 15 mL tube in 10 mL
ice-cold PBS solution, and harvest the bacteria by centrifugation at 3000 rpm (2250 Â g) for 20 min at 4
C (see Note 14).
6. Gently resuspend each pellet in 5 mL ice-cold 10% (v/v) glycerol in PBS solution, and combine resuspensions into four
15 mL centrifuge tubes (so each contains 10 mL of cell
resuspension).
7. Harvest the bacteria by centrifugation at 3000 rpm (2250 Â g)
for 20 min at 4
C (see Note 14).
8. Gently resuspend each pellet in 5 mL ice-cold 10% (v/v) glycerol in PBS solution, and combine resuspensions into two
15 mL centrifuge tubes (so each contains 10 mL of cell
resuspension).
9. Harvest the bacteria by centrifugation at 3000 rpm (2250 Â g)
for 20 min at 4
C (see Note 14).
10. Gently resuspend each pellet in 1 mL ice-cold 10% (v/v)
glycerol in PBS solution, and transfer resuspensions in prechilled 1.5 mL Eppendorf tubes.
Fig. 2 Scanning electron micrograph of the surface of a gold-microtube
membrane with tube diameter of 5 μm and density of 5 Â 10
5 cm
À2
Low-Voltage Flow-Through Electroporation Membrane and Method
47