3.2 Preparation
of Cells
1. Streak Chlamydomonas cells onto a TAP agar plate and grow
until the plates are moderately green with cells.
2. Inoculate cells into 5 mL TAP medium in 14 mL culture tube
and vortex briefly to dissociate the cells. Preculture the cells for
24 h with vigorous shaking under continuous illumination at
50 μmol photons m
–2 s
–1 .
3. Place the precultured cells on the clean bench for 5 min, transfer 2 mL of the precultured cells into 100 mL TAP medium in a
300 mL flask. Grow them on a gyratory shaker (100 rpm)
under continuous illumination at 100 μmol photons m
À2 s
À1
until the cell densities reach 1–2 Â 10
6 cells/mL,
corresponding to an optical density of 0.3–0.4 at 730 nm.
3.3 Transformation
Before electroporation, arrange all materials and supplies other
than cells on the clean bench.
1. Turn on the electroporator NEPA21 (see Note 2) and set
parameters (see Note 3) as described in Table 1 and Fig. 1.
2. Collect cells by centrifugation at 600 Â g for 5 min using a
50 mL conical tube.
3. Decant and discard the TAP medium, and gently resuspend the
cell pellet in 20 mL of prechilled Transformation buffer.
4. Centrifuge at 600 Â g for 5 min and discard the Transformation buffer.
5. Gently resuspend the pellet in 1 mL of prechilled Transformation buffer.
6. Add 2 μL of the cell suspension to the cell fix solution and
count cells using hemocytometer. Adjust the cell density at
1 Â 10
8 cells mL
À1 by adding prechilled Transformation
buffer. During the counting, place the cells at 4
C.
7. Transfer a 120 μL aliquot of the cell suspension to an electroporation cuvette for negative control.
8. Add 480 μL of the cell suspension and 12.3 μL of the aph7
00
DNA fragment solution in 1.5 mL tube, and mix well by gentle
pipetting.
9. Dispense 120 μL of the cell suspension into four electroporation cuvettes (if you want more transformants, you can increase
the volume of culture and number of cuvettes for
electroporation).
10. Chill the electroporation cuvette at 4
C for 5 min.
11. Measure electrical impedance (Ω) (see Note 4) and perform
electroporation.
158
Takashi Yamano and Hideya Fukuzawa
of Cells
1. Streak Chlamydomonas cells onto a TAP agar plate and grow
until the plates are moderately green with cells.
2. Inoculate cells into 5 mL TAP medium in 14 mL culture tube
and vortex briefly to dissociate the cells. Preculture the cells for
24 h with vigorous shaking under continuous illumination at
50 μmol photons m
–2 s
–1 .
3. Place the precultured cells on the clean bench for 5 min, transfer 2 mL of the precultured cells into 100 mL TAP medium in a
300 mL flask. Grow them on a gyratory shaker (100 rpm)
under continuous illumination at 100 μmol photons m
À2 s
À1
until the cell densities reach 1–2 Â 10
6 cells/mL,
corresponding to an optical density of 0.3–0.4 at 730 nm.
3.3 Transformation
Before electroporation, arrange all materials and supplies other
than cells on the clean bench.
1. Turn on the electroporator NEPA21 (see Note 2) and set
parameters (see Note 3) as described in Table 1 and Fig. 1.
2. Collect cells by centrifugation at 600 Â g for 5 min using a
50 mL conical tube.
3. Decant and discard the TAP medium, and gently resuspend the
cell pellet in 20 mL of prechilled Transformation buffer.
4. Centrifuge at 600 Â g for 5 min and discard the Transformation buffer.
5. Gently resuspend the pellet in 1 mL of prechilled Transformation buffer.
6. Add 2 μL of the cell suspension to the cell fix solution and
count cells using hemocytometer. Adjust the cell density at
1 Â 10
8 cells mL
À1 by adding prechilled Transformation
buffer. During the counting, place the cells at 4
C.
7. Transfer a 120 μL aliquot of the cell suspension to an electroporation cuvette for negative control.
8. Add 480 μL of the cell suspension and 12.3 μL of the aph7
00
DNA fragment solution in 1.5 mL tube, and mix well by gentle
pipetting.
9. Dispense 120 μL of the cell suspension into four electroporation cuvettes (if you want more transformants, you can increase
the volume of culture and number of cuvettes for
electroporation).
10. Chill the electroporation cuvette at 4
C for 5 min.
11. Measure electrical impedance (Ω) (see Note 4) and perform
electroporation.
158
Takashi Yamano and Hideya Fukuzawa