RI). Mix and adjust pH to 10 using 5% (w/w) sulfuric acid.
Store in a water bath at 5
C.
5. Copper foil tape, 0.25 in width. This is a copper foil with
adhesive on one side.
2.2 Electrocompetent
Bacteria Preparation
(See Note 5)
1. Lennox Luria Broth, LB (Lennox): Weigh 2 g of LB (Lennox)
microbial growth medium powder and transfer to a 200 mL
Erlenmeyer flask. Add 100 mL of water and stir to suspend.
Autoclave for 15 min at 121
C to sterilize, and allow to cool to
room temperature. Store at 4
C if not inoculated immediately
(see Note 6).
2. E. coli bacteria strain K-12 (DH5, DH10, or others) (see
Note 7).
3. Phosphate buffered saline (PBS) solution pH 7.4: Weigh
7.95 g of sodium chloride, 0.2 g of potassium chloride, 1.5 g
of sodium phosphate dibasic, and 0.2 g of potassium phosphate
monobasic, and transfer all to a 1 L volumetric flask. Dilute to
the mark with water. Agitate the flask until all salts are dissolved. Add 1 M HCl or 1 M·NaOH solution dropwise until
pH ¼ 7.4. Autoclave for 15 min at 121
C to sterilize. Allow to
cool to room temperature, then store at 4
C.
4. 10% (v/v) glycerol in PBS: Mix 10 mL of glycerol and 90 mL of
PBS solution pH 7.4 in a 150 mL Erlenmeyer flask. Autoclave
for 15 min at 121
C to sterilize. Allow to cool to room
temperature, then store at 4
C.
2.3 Electroporation
1. YO-PRO
®
-1 Iodide 1 mM in DMSO (YOPRO).
2. PBS solution pH 7.4: Prepare as in Subheading 2.2, item 3.
3. Jacketed Franz diffusion cell: diameter 15 mm, receiver chamber volume 12 mL, feed chamber volume 12 mL with O-rings
to fix the membrane (see Note 8).
4. Platinum wire, 1 mm diameter.
2.4 Analysis of
Bacteria
1. Propidium iodide (PI) 1 mM in DMSO.
2. Flow-through quartz cuvette (80 μL, 10 mm path length) for
ultraviolet–visible (UV-Vis) spectroscopy.
3. Quartz cuvette (10 mm  2 mm, 700 μL) for fluorescence
spectroscopy.
4. Clear microscope glass slide for fluorescence microscopy.
3 Methods
Carry out all procedures at room temperature, unless otherwise
specified.
Low-Voltage Flow-Through Electroporation Membrane and Method
45
Store in a water bath at 5
C.
5. Copper foil tape, 0.25 in width. This is a copper foil with
adhesive on one side.
2.2 Electrocompetent
Bacteria Preparation
(See Note 5)
1. Lennox Luria Broth, LB (Lennox): Weigh 2 g of LB (Lennox)
microbial growth medium powder and transfer to a 200 mL
Erlenmeyer flask. Add 100 mL of water and stir to suspend.
Autoclave for 15 min at 121
C to sterilize, and allow to cool to
room temperature. Store at 4
C if not inoculated immediately
(see Note 6).
2. E. coli bacteria strain K-12 (DH5, DH10, or others) (see
Note 7).
3. Phosphate buffered saline (PBS) solution pH 7.4: Weigh
7.95 g of sodium chloride, 0.2 g of potassium chloride, 1.5 g
of sodium phosphate dibasic, and 0.2 g of potassium phosphate
monobasic, and transfer all to a 1 L volumetric flask. Dilute to
the mark with water. Agitate the flask until all salts are dissolved. Add 1 M HCl or 1 M·NaOH solution dropwise until
pH ¼ 7.4. Autoclave for 15 min at 121
C to sterilize. Allow to
cool to room temperature, then store at 4
C.
4. 10% (v/v) glycerol in PBS: Mix 10 mL of glycerol and 90 mL of
PBS solution pH 7.4 in a 150 mL Erlenmeyer flask. Autoclave
for 15 min at 121
C to sterilize. Allow to cool to room
temperature, then store at 4
C.
2.3 Electroporation
1. YO-PRO
®
-1 Iodide 1 mM in DMSO (YOPRO).
2. PBS solution pH 7.4: Prepare as in Subheading 2.2, item 3.
3. Jacketed Franz diffusion cell: diameter 15 mm, receiver chamber volume 12 mL, feed chamber volume 12 mL with O-rings
to fix the membrane (see Note 8).
4. Platinum wire, 1 mm diameter.
2.4 Analysis of
Bacteria
1. Propidium iodide (PI) 1 mM in DMSO.
2. Flow-through quartz cuvette (80 μL, 10 mm path length) for
ultraviolet–visible (UV-Vis) spectroscopy.
3. Quartz cuvette (10 mm  2 mm, 700 μL) for fluorescence
spectroscopy.
4. Clear microscope glass slide for fluorescence microscopy.
3 Methods
Carry out all procedures at room temperature, unless otherwise
specified.
Low-Voltage Flow-Through Electroporation Membrane and Method
45