7. Electrocompetent E. coli are also commercially available
(DH10b, Top10 or DH5α electrocompetent cells). If these
bacteria are purchased, the procedure for making electrocompetent bacteria is not necessary.
8. Franz diffusion cells are commonly employed in the pharmaceutical industry to measure the diffusion of a drug from the
donor compartment containing the drug, through a membrane mimicking the skin, and to the receiver compartment
filled with collection medium.
9. It is important to avoid contamination between the tin chloride, silver nitrate, and gold plating solutions. Therefore, all
glassware and stir bars should be thoroughly washed before
electroless gold plating.
10. Nitric acid is corrosive and a severe irritant. Avoid contact with
skins, eyes etc.
11. E. coli can come from a freezer stock or from a single colony
from a fresh (less than 2 weeks old) LB agar plate. If from a
freezer stock, thaw the cells on ice, then pipet the solution into
the LB broth. Immediately, suck the solution back in the
pipette and expel again. Repeat three times to mix the cells
with the broth. If from a plate, use a sterile loop, pipette tip or
toothpick to pick one colony and suspend in the LB broth.
12. Because the cell population grows exponentially, the OD 600
varies exponentially with time. Measure it every hour at first,
then when it reaches 0.2, every 15–20 min. The total time
required to reach 0.4–0.5 should be around 3 h.
13. From this point on, the cultures must be kept ice-cold.
14. Dumping out all of the supernatant may result in a great loss in
cells. Therefore, first pour off about three-fourths of the supernatant. Then, remove the rest of the supernatant using a small
pipette, being careful not to suck up the cells.
15. The cells can be stored in the glycerol solution at À80
C for up
to 6 months.
16. Air bubbles would prevent the bacteria solution from passing
to the bottom receiver chamber.
17. The Pt wire in the top feed chamber should be placed as close
to the membrane as possible without touching it.
18. The number of reversibly and irreversibly electroporated bacteria can be calculated from the emission fluorescence intensity
of YOPRO using a calibration curve. To obtain this calibration
curve, lyse E. coli with a 1:4 solution of PBS and ethanol at
À20
C for 15 min, then centrifuge at 3000 rpm (2250 Â g)
for 20 min, resuspend in PBS buffer, and mix with 0.8 μM
YOPRO. Aliquot the bacteria solution into ten 1 mL
Low-Voltage Flow-Through Electroporation Membrane and Method
53
(DH10b, Top10 or DH5α electrocompetent cells). If these
bacteria are purchased, the procedure for making electrocompetent bacteria is not necessary.
8. Franz diffusion cells are commonly employed in the pharmaceutical industry to measure the diffusion of a drug from the
donor compartment containing the drug, through a membrane mimicking the skin, and to the receiver compartment
filled with collection medium.
9. It is important to avoid contamination between the tin chloride, silver nitrate, and gold plating solutions. Therefore, all
glassware and stir bars should be thoroughly washed before
electroless gold plating.
10. Nitric acid is corrosive and a severe irritant. Avoid contact with
skins, eyes etc.
11. E. coli can come from a freezer stock or from a single colony
from a fresh (less than 2 weeks old) LB agar plate. If from a
freezer stock, thaw the cells on ice, then pipet the solution into
the LB broth. Immediately, suck the solution back in the
pipette and expel again. Repeat three times to mix the cells
with the broth. If from a plate, use a sterile loop, pipette tip or
toothpick to pick one colony and suspend in the LB broth.
12. Because the cell population grows exponentially, the OD 600
varies exponentially with time. Measure it every hour at first,
then when it reaches 0.2, every 15–20 min. The total time
required to reach 0.4–0.5 should be around 3 h.
13. From this point on, the cultures must be kept ice-cold.
14. Dumping out all of the supernatant may result in a great loss in
cells. Therefore, first pour off about three-fourths of the supernatant. Then, remove the rest of the supernatant using a small
pipette, being careful not to suck up the cells.
15. The cells can be stored in the glycerol solution at À80
C for up
to 6 months.
16. Air bubbles would prevent the bacteria solution from passing
to the bottom receiver chamber.
17. The Pt wire in the top feed chamber should be placed as close
to the membrane as possible without touching it.
18. The number of reversibly and irreversibly electroporated bacteria can be calculated from the emission fluorescence intensity
of YOPRO using a calibration curve. To obtain this calibration
curve, lyse E. coli with a 1:4 solution of PBS and ethanol at
À20
C for 15 min, then centrifuge at 3000 rpm (2250 Â g)
for 20 min, resuspend in PBS buffer, and mix with 0.8 μM
YOPRO. Aliquot the bacteria solution into ten 1 mL
Low-Voltage Flow-Through Electroporation Membrane and Method
53