Before electroporation, the cell suspension needs to be mixed
with the plasmid, and then the mixture is transferred to the electric
shock cuvette. The voltage and capacitance are set and the electric
shock cup is inserted into the electroporator. Electroporation
works by transmitting kilovolts through 1–2 mm thick suspension
cells in the electric shock cuvette (1.0–1.5 kV, 0.5–7.5 kV/cm).
Cells should then be handled carefully until they have a chance to
divide and produce new cells containing regenerated plasmids. This
process is about ten times more efficient than chemical conversion.
2 Materials
2.1 Apparatus
Electroporation apparatus Gene Pulser Xcell (Bio-Rad) (see
Note 1).
Light incubator (see Note 2).
pH meter.
High-speed refrigerated centrifuge.
2.2 Buffer
Preparation
1. Hypertonic solution: 0.2 M mannitol, 0.2 M sorbitol, sterilized at 115
C for 30 min and then stored at 4
C.
2. Electroporation buffer: 0.2 M mannitol, 0.2 M sorbitol,
0.08 M KCl, 0.005 M CaCl 2 , 0.01 M HEPES (pH 7.2),
sterilized at 115
C for 30 min and then stored at 4
C.
3. Endo medium.
4. BBM medium containing 1 g/L glucose.
5. SE solid medium (1.5% agar) containing G418 (25 mg/L) (see
Note 3).
6. Commercially available Salmon sperm DNA is used from a
stock solution (10 mg/mL).
3 Methods
Electrotransformation is described on freshwater-growing species
Chlorella pyrenoidosa [7].
1. Inoculate 10% Chlorella pyrenoidosa into the Endo medium.
Culture at 30
C and with a rotary shaker (150 rpm) under low
light condition in a flask (~100 μmol/m
2 /s).
2. Collect the algal cells at logarithmic growth stage and centrifuge 1 mL at 4
C for 3 min at 10,000 Â g (see Note 4).
3. Suck out the supernatant carefully and added with 1 mL
Hypertonic solution (see Note 5).
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Liang Ji and Jianhua Fan
with the plasmid, and then the mixture is transferred to the electric
shock cuvette. The voltage and capacitance are set and the electric
shock cup is inserted into the electroporator. Electroporation
works by transmitting kilovolts through 1–2 mm thick suspension
cells in the electric shock cuvette (1.0–1.5 kV, 0.5–7.5 kV/cm).
Cells should then be handled carefully until they have a chance to
divide and produce new cells containing regenerated plasmids. This
process is about ten times more efficient than chemical conversion.
2 Materials
2.1 Apparatus
Electroporation apparatus Gene Pulser Xcell (Bio-Rad) (see
Note 1).
Light incubator (see Note 2).
pH meter.
High-speed refrigerated centrifuge.
2.2 Buffer
Preparation
1. Hypertonic solution: 0.2 M mannitol, 0.2 M sorbitol, sterilized at 115
C for 30 min and then stored at 4
C.
2. Electroporation buffer: 0.2 M mannitol, 0.2 M sorbitol,
0.08 M KCl, 0.005 M CaCl 2 , 0.01 M HEPES (pH 7.2),
sterilized at 115
C for 30 min and then stored at 4
C.
3. Endo medium.
4. BBM medium containing 1 g/L glucose.
5. SE solid medium (1.5% agar) containing G418 (25 mg/L) (see
Note 3).
6. Commercially available Salmon sperm DNA is used from a
stock solution (10 mg/mL).
3 Methods
Electrotransformation is described on freshwater-growing species
Chlorella pyrenoidosa [7].
1. Inoculate 10% Chlorella pyrenoidosa into the Endo medium.
Culture at 30
C and with a rotary shaker (150 rpm) under low
light condition in a flask (~100 μmol/m
2 /s).
2. Collect the algal cells at logarithmic growth stage and centrifuge 1 mL at 4
C for 3 min at 10,000 Â g (see Note 4).
3. Suck out the supernatant carefully and added with 1 mL
Hypertonic solution (see Note 5).
182
Liang Ji and Jianhua Fan