achieve higher transformation efficiency compared with microprojectile bombardment, while the latter applied a multipulse electroporation system dependent on a special apparatus.
We developed a simple method for high efficiency nuclear
transformation of P. tricornutum by electroporation. The procedure was based on a universal electroporation protocol and the
successful transformation of P. tricornutum requires linearized plasmid DNA with salmon sperm DNA as carrier.
2 Materials
Prepare all solutions using ultrapure water (deionized water that
was purified to attain a conductivity of 18 MΩ cm at 25
C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise).
2.1 Strain
and Culture Medium
1. Strain: Phaeodactylum tricornutum Bohlin (CCMP2561) was
obtained from the culture collection of the Provasoli-Guillard
National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, USA.
2. Medium: P. tricornutum were grown axenically in artificial
seawater (ASW) [17] enriched with f/2 nutrients (main components: 0.88 mM NaNO 3 , 36.3 μM NaH 2 PO 4 ; micronutrients: 0.08 μM ZnSO 4 , 0.9 μM MnCl 2 , 0.03 μM Na 2 MoO 4 ,
0.05 μM CoCl 2 , 0.04 μM CuSO 4 , 11.7 μM FeCl 3 , 11.7 μM
EDTA; vitamin: 0.5 μg/L cyanocobalamin, 0.5 μg/L biotin,
100 μg/L thiamine–HCl) according to Guillard [18]. For
growth on solid media, cultures were grown on 50% ASW
containing f/2 nutrients, 1.2% agar.
3. Working Solutions: 375 mM sorbitol solution:
2.2 Reagents
1. 10 mg/mL salmon sperm DNA is frozen-stored at À20
C and
denatured by boiling for 1~5 min before use.
2. Zeocin™.
3. Restriction enzymes ScaI.
4. Plasmid extraction kit and DNA purification kit.
2.3 Equipment
1. Centrifuge (4
C, up to 1500 Â g).
2. Micropipettes (variable volume).
3. Water bath (100
C).
4. Cell electroporation system (Bio-Rad Gene Pulser X, 2-mm
electroporation cuvettes).
164
Hanhua Hu and Yufang Pan
We developed a simple method for high efficiency nuclear
transformation of P. tricornutum by electroporation. The procedure was based on a universal electroporation protocol and the
successful transformation of P. tricornutum requires linearized plasmid DNA with salmon sperm DNA as carrier.
2 Materials
Prepare all solutions using ultrapure water (deionized water that
was purified to attain a conductivity of 18 MΩ cm at 25
C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise).
2.1 Strain
and Culture Medium
1. Strain: Phaeodactylum tricornutum Bohlin (CCMP2561) was
obtained from the culture collection of the Provasoli-Guillard
National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences, USA.
2. Medium: P. tricornutum were grown axenically in artificial
seawater (ASW) [17] enriched with f/2 nutrients (main components: 0.88 mM NaNO 3 , 36.3 μM NaH 2 PO 4 ; micronutrients: 0.08 μM ZnSO 4 , 0.9 μM MnCl 2 , 0.03 μM Na 2 MoO 4 ,
0.05 μM CoCl 2 , 0.04 μM CuSO 4 , 11.7 μM FeCl 3 , 11.7 μM
EDTA; vitamin: 0.5 μg/L cyanocobalamin, 0.5 μg/L biotin,
100 μg/L thiamine–HCl) according to Guillard [18]. For
growth on solid media, cultures were grown on 50% ASW
containing f/2 nutrients, 1.2% agar.
3. Working Solutions: 375 mM sorbitol solution:
2.2 Reagents
1. 10 mg/mL salmon sperm DNA is frozen-stored at À20
C and
denatured by boiling for 1~5 min before use.
2. Zeocin™.
3. Restriction enzymes ScaI.
4. Plasmid extraction kit and DNA purification kit.
2.3 Equipment
1. Centrifuge (4
C, up to 1500 Â g).
2. Micropipettes (variable volume).
3. Water bath (100
C).
4. Cell electroporation system (Bio-Rad Gene Pulser X, 2-mm
electroporation cuvettes).
164
Hanhua Hu and Yufang Pan