3. The harvested cells are washed six times with ice-cold, filtersterilized 375 mM sorbitol, and after the washing step, the cells
are finally resuspended with 0.2 mL of 375 mM sorbitol (see
Note 3).
3.2 Preparation
of Cell and DNA
Reaction Mixture
1. For each electroporation reaction, 0.2 mL of cells resuspended
in sorbitol is taken.
2. The desired recombinant plasmid is linearized by using specific
restriction enzymes and purified by using DNA fragment purification as per the manufacturer’s instruction.
3. Thereafter, we electroporate the eGFP gene cloned into the
expression vector pNa03 (pNa03-eGFP) into N. oceanica cells.
The recombinant vector harbors the Sh-ble gene which confers
resistance to Zeocin to screen the transformants. The linearized
plasmid (3–5 μg) is used for electroporation (see Note 4).
4. Each electroporation method should be performed with one
control reaction, which contains only salmon sperm DNA and
no other DNA molecules.
3.3 Electroporation
1. The cells, salmon sperm DNA (30–50 μg) and recombinant
DNA are mixed and transferred to a 2 mm cuvette and kept in
ice for 10 min prior to electroporation.
2. After ice incubation, the cuvettes are completely wiped by
tissue paper and subsequently, electroporated.
3. Nannochloropsis oceanica electroporation is performed by using
the following parameters: Voltage 2200 V, 50 μF and resistance
at 600 Ω. For handling the electroporator effectively, instructions from the manual provided by the manufacturer should be
read and carefully observed.
4. After electroporation, the cells are transferred to 5 mL of IMK
liquid medium and incubated in dark at 25 Æ 1
C for 24 h (see
Note 5).
3.4 Plating
into Antibiotic Medium
1. Following the 24-h dark incubation, the cells are harvested by
centrifugation at 4000 Â g for 5 min.
2. Subsequently, ~100 μL of cells from both recombinant DNA
introduced and control cells is collected and individually spread
onto the IMK agar plates supplemented with an appropriate
antibiotic.
3. Emerging independent colonies with uniform phenotypes are
further
screened
by
physiological
and
molecular
analyses (Fig. 1).
4. Independent colonies are further subcultured into the IMK
liquid medium supplemented with Zeocin, and the successful
expression of the target gene is further confirmed by semi
quantitative RT-PCR (see Note 6).
Electroporation of Nannochloropsis
177
are finally resuspended with 0.2 mL of 375 mM sorbitol (see
Note 3).
3.2 Preparation
of Cell and DNA
Reaction Mixture
1. For each electroporation reaction, 0.2 mL of cells resuspended
in sorbitol is taken.
2. The desired recombinant plasmid is linearized by using specific
restriction enzymes and purified by using DNA fragment purification as per the manufacturer’s instruction.
3. Thereafter, we electroporate the eGFP gene cloned into the
expression vector pNa03 (pNa03-eGFP) into N. oceanica cells.
The recombinant vector harbors the Sh-ble gene which confers
resistance to Zeocin to screen the transformants. The linearized
plasmid (3–5 μg) is used for electroporation (see Note 4).
4. Each electroporation method should be performed with one
control reaction, which contains only salmon sperm DNA and
no other DNA molecules.
3.3 Electroporation
1. The cells, salmon sperm DNA (30–50 μg) and recombinant
DNA are mixed and transferred to a 2 mm cuvette and kept in
ice for 10 min prior to electroporation.
2. After ice incubation, the cuvettes are completely wiped by
tissue paper and subsequently, electroporated.
3. Nannochloropsis oceanica electroporation is performed by using
the following parameters: Voltage 2200 V, 50 μF and resistance
at 600 Ω. For handling the electroporator effectively, instructions from the manual provided by the manufacturer should be
read and carefully observed.
4. After electroporation, the cells are transferred to 5 mL of IMK
liquid medium and incubated in dark at 25 Æ 1
C for 24 h (see
Note 5).
3.4 Plating
into Antibiotic Medium
1. Following the 24-h dark incubation, the cells are harvested by
centrifugation at 4000 Â g for 5 min.
2. Subsequently, ~100 μL of cells from both recombinant DNA
introduced and control cells is collected and individually spread
onto the IMK agar plates supplemented with an appropriate
antibiotic.
3. Emerging independent colonies with uniform phenotypes are
further
screened
by
physiological
and
molecular
analyses (Fig. 1).
4. Independent colonies are further subcultured into the IMK
liquid medium supplemented with Zeocin, and the successful
expression of the target gene is further confirmed by semi
quantitative RT-PCR (see Note 6).
Electroporation of Nannochloropsis
177