15. Plate the cell suspension onto TAP agar plate containing
hygromycin B. Each suspension originating from a single electroporation cuvette is dispensed onto four selective plates.
16. Place the plates under continuous light at 100 μmol photons
m
À2 s
À1 for 7–10 days (see Note 5).
4 Notes
1. In this method, a Chlamydomonas reinhardtii strain C-9 is used
as a representative of cell-walled wild-type cells. C-9 is available
from National Institute for Environmental Studies, Japan, as
strain NIES-2235.
2. In contrast to other electroporators, such as Gene Pulser series
(Bio-Rad, USA) or ECM series (BTX, USA), NEPA21 has
three-step multiple electroporation pulses, resulting in higher
transformation efficiency and lower damage to the cells. The
first pulse is a poring pulse (Pp) with high voltage and short
pulse length. The second pulse consists of multiple transfer
pulses (Tp) with low voltage and long pulse length for delivering exogenous DNA into cells. The third pulse is polarityexchanged Tp for the efficient delivery of DNA molecules
into cells.
3. Six parameters, voltage (V), pulse length (ms), pulse interval
(ms), number of pulses, decay rates (%), and polarity, are set for
respective Pp and Tp. In addition, values of electrical impedance between the electrodes (Ω), actual voltage (V), current
(A), and energy (J) can be measured.
4. The measured value of the electrical impedance is approximately 0.2 kΩ in the cell conditions described above.
5. Typically, 500–1,000 hygromycin-resistant colonies appear per
plate. With increasing DNA concentrations, the number of
transformants increased. However, the inserted copy number
of aph7
00 cassette could be increased.
Acknowledgments
This work was partly supported by Advanced Low Carbon Technology Research and Development Program (ALCA,
JPMJAL1105) and by Grants-in-Aid for Scientific Research from
the Japan Society for the Promotion of Science (25120714 and
16K07399).
160
Takashi Yamano and Hideya Fukuzawa
hygromycin B. Each suspension originating from a single electroporation cuvette is dispensed onto four selective plates.
16. Place the plates under continuous light at 100 μmol photons
m
À2 s
À1 for 7–10 days (see Note 5).
4 Notes
1. In this method, a Chlamydomonas reinhardtii strain C-9 is used
as a representative of cell-walled wild-type cells. C-9 is available
from National Institute for Environmental Studies, Japan, as
strain NIES-2235.
2. In contrast to other electroporators, such as Gene Pulser series
(Bio-Rad, USA) or ECM series (BTX, USA), NEPA21 has
three-step multiple electroporation pulses, resulting in higher
transformation efficiency and lower damage to the cells. The
first pulse is a poring pulse (Pp) with high voltage and short
pulse length. The second pulse consists of multiple transfer
pulses (Tp) with low voltage and long pulse length for delivering exogenous DNA into cells. The third pulse is polarityexchanged Tp for the efficient delivery of DNA molecules
into cells.
3. Six parameters, voltage (V), pulse length (ms), pulse interval
(ms), number of pulses, decay rates (%), and polarity, are set for
respective Pp and Tp. In addition, values of electrical impedance between the electrodes (Ω), actual voltage (V), current
(A), and energy (J) can be measured.
4. The measured value of the electrical impedance is approximately 0.2 kΩ in the cell conditions described above.
5. Typically, 500–1,000 hygromycin-resistant colonies appear per
plate. With increasing DNA concentrations, the number of
transformants increased. However, the inserted copy number
of aph7
00 cassette could be increased.
Acknowledgments
This work was partly supported by Advanced Low Carbon Technology Research and Development Program (ALCA,
JPMJAL1105) and by Grants-in-Aid for Scientific Research from
the Japan Society for the Promotion of Science (25120714 and
16K07399).
160
Takashi Yamano and Hideya Fukuzawa