3 Methods
Carry out all procedures at room temperature in a sterile environment, unless otherwise specified.
1. Prepare plasmid DNA: isolate plasmid DNA from overnightcultured Escherichia coli cells containing plasmid pPha-T1 [4]
according to the operation steps of the plasmid extraction kit.
Linearize 8–10 μg plasmid DNA by ScaI (see Note 1) digestion
at 37
C according to the instruction and then purify the
linearized plasmid DNA by DNA purification kit (see Note 2).
2. P. tricornutum growth conditions: 22
C under cool white
fluorescent light (75 μmol photons/m
2 s) with a photoperiod
of 12 h light and 12 h dark in 250 mL Erlenmeyer flask. Cells
on solid media were grown at 22
C and continuously illuminated with 75 μmol photons/m
2 s.
3. Prepare the 375 mM Sorbitol solution: Weigh 6.83 g D-sorbitol and transfer it to a 100 mL bottle. Add about 80 mL
ultrapure water to the bottle and stir until the sorbitol has
dissolved completely (see Note 3). Bring up to 100 mL and
sterilize through a 0.2 μm membrane filter using a water jet
pump. Store sorbitol solution at 4
C for further use.
4. Centrifuge the mid-logarithmic phase P. tricornutum culture
(cell density is about 4~5 Â 10
6 cells/mL) at 1500 Â g for
10 min at 4
C in a 50 mL Falcon tube. A total of 2 Â 10
8 cells
are needed in one electroporation reaction.
5. Perform a washing step: Resuspend the resulting pellet gently
but thoroughly in 1 mL of ice-cold 375 mM sorbitol solution
(see Note 4). Centrifuge the cells at 1500 Â g for 4 min at 4
C
in a 1.5 mL Eppendorf tube.
6. Repeat the washing step twice.
7. Resuspend the resulting pellet in 100 μL 375 mM sorbitol.
Add 4 μg linearized plasmid DNA (see Note 5) and 40 μg
salmon sperm DNA (10 mg/mL, denatured by boiling for
1~5 min before use). Mix gently but thoroughly and incubate
on ice for 30 min.
8. Transfer the cell suspension into a 2-mm electroporation
cuvette. The electroporation system is adjusted to exponential
decay, 0.5 kV voltage, 25 μF capacitance, and 400 Ω shunt
resistance.
9. After electroporation, immediately transfer the cells to 15 mL
conical Falcon tubes containing 10 mL f/2 medium. Then
incubate them at 22
C in low light (~30 μmol photons/
m
2 s) overnight without shaking.
Phaeodactylum tricornutum
165
Carry out all procedures at room temperature in a sterile environment, unless otherwise specified.
1. Prepare plasmid DNA: isolate plasmid DNA from overnightcultured Escherichia coli cells containing plasmid pPha-T1 [4]
according to the operation steps of the plasmid extraction kit.
Linearize 8–10 μg plasmid DNA by ScaI (see Note 1) digestion
at 37
C according to the instruction and then purify the
linearized plasmid DNA by DNA purification kit (see Note 2).
2. P. tricornutum growth conditions: 22
C under cool white
fluorescent light (75 μmol photons/m
2 s) with a photoperiod
of 12 h light and 12 h dark in 250 mL Erlenmeyer flask. Cells
on solid media were grown at 22
C and continuously illuminated with 75 μmol photons/m
2 s.
3. Prepare the 375 mM Sorbitol solution: Weigh 6.83 g D-sorbitol and transfer it to a 100 mL bottle. Add about 80 mL
ultrapure water to the bottle and stir until the sorbitol has
dissolved completely (see Note 3). Bring up to 100 mL and
sterilize through a 0.2 μm membrane filter using a water jet
pump. Store sorbitol solution at 4
C for further use.
4. Centrifuge the mid-logarithmic phase P. tricornutum culture
(cell density is about 4~5 Â 10
6 cells/mL) at 1500 Â g for
10 min at 4
C in a 50 mL Falcon tube. A total of 2 Â 10
8 cells
are needed in one electroporation reaction.
5. Perform a washing step: Resuspend the resulting pellet gently
but thoroughly in 1 mL of ice-cold 375 mM sorbitol solution
(see Note 4). Centrifuge the cells at 1500 Â g for 4 min at 4
C
in a 1.5 mL Eppendorf tube.
6. Repeat the washing step twice.
7. Resuspend the resulting pellet in 100 μL 375 mM sorbitol.
Add 4 μg linearized plasmid DNA (see Note 5) and 40 μg
salmon sperm DNA (10 mg/mL, denatured by boiling for
1~5 min before use). Mix gently but thoroughly and incubate
on ice for 30 min.
8. Transfer the cell suspension into a 2-mm electroporation
cuvette. The electroporation system is adjusted to exponential
decay, 0.5 kV voltage, 25 μF capacitance, and 400 Ω shunt
resistance.
9. After electroporation, immediately transfer the cells to 15 mL
conical Falcon tubes containing 10 mL f/2 medium. Then
incubate them at 22
C in low light (~30 μmol photons/
m
2 s) overnight without shaking.
Phaeodactylum tricornutum
165