11. Inoculate the Agrobacterium colony in 2 mL of LB medium
containing appropriate antibiotics for shaking incubation at
28
C with 180–220 rpm overnight.
12. Extract the plasmid DNA from each Agrobacterium culture
and retransform plasmid DNA into chemical-competent
E. coli DH5α cells.
13. After heat-shock transformation, add 1 mL of LB medium to
each 1.5-mL microcentrifuge tube and incubate with agitation
for 1 h at 37
C.
14. Remove supernatant and plate 100 μL of the suspension on
fresh LB agar plates containing appropriate antibiotics. Incubate the plate overnight at 37
C.
15. Pick two E. coli colonies from each plate into 3 mL of LB
medium containing appropriate antibiotics for shaking incubation with 180–220 rpm overnight at 37
C.
16. Extract the plasmid DNA from each E. coli culture and check
the restriction digestion map of the plasmid DNA for both
colonies (see Note 2).
17. After confirming the restriction digestion results by electrophoresis, inoculate the positive Agrobacterium colony into
2 mL of LB medium containing appropriate antibiotics for
shaking incubation with 180–220 rpm overnight at 28
C.
18. Prepare glycerol stocks by mixing 500 μL Agrobacterium culture with 500 μL sterile 50% glycerol in 2-mL cryogenic tube.
Flash freeze the tube in liquid N 2 and store at À80
C. For
frequent usage, Agrobacterium strains could be stored at 4
C
on LB agar plates with appropriate antibiotics if transferred
biweekly to a new plate.
3.2 Growing
of Nicotiana
Benthamiana
1. For seeds sterilization, let N. benthamiana seeds be immersed
with 1 mL of seed sterilization solution in 1.5-mL tube. Vortex
for 30 s and remove the solution as much as possible.
2. Wash the seeds with 1 mL of sterile dH 2 O and vortex for 15 s
and repeat four times.
3. Remove the sterile dH 2 O as much as possible.
4. Add 200 μL of 95% EtOH to the tube and immediately transfer
the solution containing seeds directly unto filter paper in the
sterile hood using a trimmed 200-μL tip.
5. After seeds completely dry, hold the filter paper and spread
seeds evenly and slowly on top of 0.5 MS agar.
6. Wrap up the 0.5 MS agar plate with surgical tape. Place the
plate for 48 h at 4
C in the dark. Move the plate to growth
chamber for 7–10 days at 24
C for seedling growth.
7. Prepare pots with moist growing matrix.
330
Tzu-Yin Liu
containing appropriate antibiotics for shaking incubation at
28
C with 180–220 rpm overnight.
12. Extract the plasmid DNA from each Agrobacterium culture
and retransform plasmid DNA into chemical-competent
E. coli DH5α cells.
13. After heat-shock transformation, add 1 mL of LB medium to
each 1.5-mL microcentrifuge tube and incubate with agitation
for 1 h at 37
C.
14. Remove supernatant and plate 100 μL of the suspension on
fresh LB agar plates containing appropriate antibiotics. Incubate the plate overnight at 37
C.
15. Pick two E. coli colonies from each plate into 3 mL of LB
medium containing appropriate antibiotics for shaking incubation with 180–220 rpm overnight at 37
C.
16. Extract the plasmid DNA from each E. coli culture and check
the restriction digestion map of the plasmid DNA for both
colonies (see Note 2).
17. After confirming the restriction digestion results by electrophoresis, inoculate the positive Agrobacterium colony into
2 mL of LB medium containing appropriate antibiotics for
shaking incubation with 180–220 rpm overnight at 28
C.
18. Prepare glycerol stocks by mixing 500 μL Agrobacterium culture with 500 μL sterile 50% glycerol in 2-mL cryogenic tube.
Flash freeze the tube in liquid N 2 and store at À80
C. For
frequent usage, Agrobacterium strains could be stored at 4
C
on LB agar plates with appropriate antibiotics if transferred
biweekly to a new plate.
3.2 Growing
of Nicotiana
Benthamiana
1. For seeds sterilization, let N. benthamiana seeds be immersed
with 1 mL of seed sterilization solution in 1.5-mL tube. Vortex
for 30 s and remove the solution as much as possible.
2. Wash the seeds with 1 mL of sterile dH 2 O and vortex for 15 s
and repeat four times.
3. Remove the sterile dH 2 O as much as possible.
4. Add 200 μL of 95% EtOH to the tube and immediately transfer
the solution containing seeds directly unto filter paper in the
sterile hood using a trimmed 200-μL tip.
5. After seeds completely dry, hold the filter paper and spread
seeds evenly and slowly on top of 0.5 MS agar.
6. Wrap up the 0.5 MS agar plate with surgical tape. Place the
plate for 48 h at 4
C in the dark. Move the plate to growth
chamber for 7–10 days at 24
C for seedling growth.
7. Prepare pots with moist growing matrix.
330
Tzu-Yin Liu