2. Add 12 mL of a solution of 0.8 μM YOPRO in ice-cold PBS
pH 7.4 and a stir bar to the receiver chamber.
3. Insert a capillary tube through the sampling port of the receiver
chamber, and seal the port with Parafilm.
4. Connect the capillary tube to the flow-through UV-Vis cuvette
(Fig. 3). Insert the cuvette into a UV-Vis spectrophotometer
and use a pump to pump the receiver solution through the
cuvette.
Install the Franz diffusion cell top feed chamber (Fig. 3):
5. With the bottom receiver chamber filled with the YOPRO
solution, place the gold-microtube membrane with the gold
film facing down between the O-rings that join the bottom and
top chambers of the Franz diffusion cell. Make sure that there
are no air bubbles on the bottom of the membrane where it
contacts the YOPRO solution (see Note 16).
6. Position the feed chamber on top of the membrane, and hold
the two chambers and the membrane together using a clamp
(not shown in Fig. 3).
7. Connect one electrode of a square-wave or pulse generator to
the copper tape attached to the membrane (Figs. 1 and 3), and
the other electrode to a platinum wire in the feed chamber (see
Note 17).
8. Add 3 mL of the solution containing the electrocompetent
bacteria and 0.8 μM YOPRO in ice-cold PBS pH 7.4 to the
feed chamber.
Start flow-through electroporation:
9. After adding the bacteria solution, immediately turn on the
pulse generator. Apply À4 V pulses of 30 ms duration every
250 ms (frequency 3.6 Hz).
10. Turn on the pump to pull the bacteria solution through the
membrane to the receiver chamber and then to the UV-Vis
spectrophotometer at a flow rate of 2 μL/s.
3.4 Analysis of
Bacteria
First method of analysis by flow-through OD 600 and fluorescence
intensity measurements:
1. Measure the OD 600 continuously during electroporation using
the flow-through UV-Vis cuvette and spectrophotometer
(Fig. 4a). Use the OD 600 to determine the concentration of
bacteria that passed through the membrane (OD 600 of
1.0 ¼ 8 Â 10
8
E. coli/mL).
2. At the same time, collect volumes of 600 μL of the solution
exiting the UV-Vis spectrophotometer every 5 min in 1 mL
Eppendorf tubes.
3. Place all the tubes on ice for at least 30 min.
Low-Voltage Flow-Through Electroporation Membrane and Method
49
pH 7.4 and a stir bar to the receiver chamber.
3. Insert a capillary tube through the sampling port of the receiver
chamber, and seal the port with Parafilm.
4. Connect the capillary tube to the flow-through UV-Vis cuvette
(Fig. 3). Insert the cuvette into a UV-Vis spectrophotometer
and use a pump to pump the receiver solution through the
cuvette.
Install the Franz diffusion cell top feed chamber (Fig. 3):
5. With the bottom receiver chamber filled with the YOPRO
solution, place the gold-microtube membrane with the gold
film facing down between the O-rings that join the bottom and
top chambers of the Franz diffusion cell. Make sure that there
are no air bubbles on the bottom of the membrane where it
contacts the YOPRO solution (see Note 16).
6. Position the feed chamber on top of the membrane, and hold
the two chambers and the membrane together using a clamp
(not shown in Fig. 3).
7. Connect one electrode of a square-wave or pulse generator to
the copper tape attached to the membrane (Figs. 1 and 3), and
the other electrode to a platinum wire in the feed chamber (see
Note 17).
8. Add 3 mL of the solution containing the electrocompetent
bacteria and 0.8 μM YOPRO in ice-cold PBS pH 7.4 to the
feed chamber.
Start flow-through electroporation:
9. After adding the bacteria solution, immediately turn on the
pulse generator. Apply À4 V pulses of 30 ms duration every
250 ms (frequency 3.6 Hz).
10. Turn on the pump to pull the bacteria solution through the
membrane to the receiver chamber and then to the UV-Vis
spectrophotometer at a flow rate of 2 μL/s.
3.4 Analysis of
Bacteria
First method of analysis by flow-through OD 600 and fluorescence
intensity measurements:
1. Measure the OD 600 continuously during electroporation using
the flow-through UV-Vis cuvette and spectrophotometer
(Fig. 4a). Use the OD 600 to determine the concentration of
bacteria that passed through the membrane (OD 600 of
1.0 ¼ 8 Â 10
8
E. coli/mL).
2. At the same time, collect volumes of 600 μL of the solution
exiting the UV-Vis spectrophotometer every 5 min in 1 mL
Eppendorf tubes.
3. Place all the tubes on ice for at least 30 min.
Low-Voltage Flow-Through Electroporation Membrane and Method
49