4. Mix with pipette tips gently.
5. Keep in an ice bath for 40 min (see Note 6).
6. Centrifuge the mixture at 4
C for 3 min at 10,000 Â g.
7. Collect the algal cells by sucking out the supernatant carefully.
8. Add 1 mL Electroporation buffer to the cell pellet and mix with
pipette tips gently.
9. Study cell density by hemacytometry.
10. Adjust the cell density by Electroporation buffer to 5 Â 10
7
/mL.
11. Add 30 μg/mL plasmids (see Note 7), and 150 μg/mL salmon
sperm DNA (see Note 8), and mix gently.
12. Add 100 μL mixture into a 2 mm electric shock cuvette.
13. Keep in an ice bath for 5 min (see Note 6).
14. Set the appropriate parameters (660 V pulse voltage; 3.5 ms
pulse width) to carry on the electric shock (see Note 9).
15. Place the cells in the cuvette on ice for 10 min after the electric
shock is delivered.
16. Add 200 μL BBM medium containing 1 g/L glucose.
17. Store at 30
C for 1 h (see Note 10).
18. Suck out the culture medium carefully with a pipette and
transfer to a centrifugal tube.
19. Collect the algal cells by centrifuging at 4
C for 2 min at
16,000 Â g.
20. Add 150 μL BBM medium containing 1 g/L glucose.
21. Recovery is obtained by culturing in sterile 96-well plates for
24 h (see Note 11).
22. Take out all of the algal cells and spray on the SE solid medium
(1.5% agar) containing G418 (25 mg/L).
23. Culture in dark condition at 30
C for one night firstly before
culturing in the light–dark cycle of 60 μmol/m
2 /s and 16 h/
8 h. The single algae could grow out about a week later.
4 Notes
1. BTX-630 can be used.
2. It was a SPX-300 from Shanghai Bo Xun Industrial Co., Ltd.,
in our assay.
3. Choose the appropriate medium according to the research
object.
4. In the logarithmic growth phase, the cells proliferate rapidly
and the cell walls are relaxed. There are many cells in mitotic
Electrotransformation of Chlorella Species
183
5. Keep in an ice bath for 40 min (see Note 6).
6. Centrifuge the mixture at 4
C for 3 min at 10,000 Â g.
7. Collect the algal cells by sucking out the supernatant carefully.
8. Add 1 mL Electroporation buffer to the cell pellet and mix with
pipette tips gently.
9. Study cell density by hemacytometry.
10. Adjust the cell density by Electroporation buffer to 5 Â 10
7
/mL.
11. Add 30 μg/mL plasmids (see Note 7), and 150 μg/mL salmon
sperm DNA (see Note 8), and mix gently.
12. Add 100 μL mixture into a 2 mm electric shock cuvette.
13. Keep in an ice bath for 5 min (see Note 6).
14. Set the appropriate parameters (660 V pulse voltage; 3.5 ms
pulse width) to carry on the electric shock (see Note 9).
15. Place the cells in the cuvette on ice for 10 min after the electric
shock is delivered.
16. Add 200 μL BBM medium containing 1 g/L glucose.
17. Store at 30
C for 1 h (see Note 10).
18. Suck out the culture medium carefully with a pipette and
transfer to a centrifugal tube.
19. Collect the algal cells by centrifuging at 4
C for 2 min at
16,000 Â g.
20. Add 150 μL BBM medium containing 1 g/L glucose.
21. Recovery is obtained by culturing in sterile 96-well plates for
24 h (see Note 11).
22. Take out all of the algal cells and spray on the SE solid medium
(1.5% agar) containing G418 (25 mg/L).
23. Culture in dark condition at 30
C for one night firstly before
culturing in the light–dark cycle of 60 μmol/m
2 /s and 16 h/
8 h. The single algae could grow out about a week later.
4 Notes
1. BTX-630 can be used.
2. It was a SPX-300 from Shanghai Bo Xun Industrial Co., Ltd.,
in our assay.
3. Choose the appropriate medium according to the research
object.
4. In the logarithmic growth phase, the cells proliferate rapidly
and the cell walls are relaxed. There are many cells in mitotic
Electrotransformation of Chlorella Species
183