17. Sterile hood.
18. Greenhouse and/or plant growth device to maintain stable
growth conditions to enable light intensities of 50–120 μE
m
À2 s
À1 under long day (16 h light/8 h dark) conditions.
19. Freezer (À20 and À80
C).
20. Fridge and/or cold room (4
C).
2.6 Antibiotics
and Reagent Stock
Solution
1. 50 mg/mL Rifampicin stock in dimethyl sulfoxide (DMSO).
Store aliquots at À20
C. Working concentration 5 μg/mL.
2. 50 mg/mL Kanamycin stock in H 2 O. Store aliquots at
À20
C. Working concentration 50 μg/mL.
3. 50 mg/mL Gentamycin stock in H 2 O. Store aliquots at
À20
C. Working concentration 50 μg/mL.
4. 150 mM 3,5-dimethoxy-4-hydroxyacetophenone (Acetosyringone) stock in DMSO. Store aliquots at À20
C.
5. 10 mg/ml (36.7 mM) β-Estradiol stock in EtOH. Store aliquots at À20
C. Working concentration 2.5–10 μg/mL.
3 Methods
3.1 Transformation
and Culture
of Agrobacterium
1. Prepare for each transformation one 1.5-mL tube.
2. Thaw for each transformation one 100 μL aliquot of Agrobacterium competent cells (stored at À80
C) on ice.
3. Add 1 μg of plasmid DNA to each tube and mix by tapping
with your index finger and keep them on ice for 3 min.
4. Transfer the tubes to liquid nitrogen to completely freeze the
samples for 3 min
5. Transfer the tubes to 37
C water bath and incubate for 3 min.
6. Immediately after the above free-thaw transformation, add
1 mL of LB medium to each 1.5-mL microcentrifuge tube.
7. Gently rotate the tubes for 4 h at 28
C (or room temperature)
using rotating mixer.
8. Spin the tubes at 1500 Â g for 5 min to get pellet cells.
9. Remove supernatant and plate 100 μL of the suspension on
fresh LB agar plates containing appropriate antibiotics. Incubate the plate for 2–3 days at 28
C until several big colonies
become visible.
10. Pick one colony from each plate and re-streak on a new LB agar
plate containing appropriate antibiotics. Incubate the plate
overnight at 28
C and keep it for the future use.
Tripartite Split-sfGFP Assay
329
18. Greenhouse and/or plant growth device to maintain stable
growth conditions to enable light intensities of 50–120 μE
m
À2 s
À1 under long day (16 h light/8 h dark) conditions.
19. Freezer (À20 and À80
C).
20. Fridge and/or cold room (4
C).
2.6 Antibiotics
and Reagent Stock
Solution
1. 50 mg/mL Rifampicin stock in dimethyl sulfoxide (DMSO).
Store aliquots at À20
C. Working concentration 5 μg/mL.
2. 50 mg/mL Kanamycin stock in H 2 O. Store aliquots at
À20
C. Working concentration 50 μg/mL.
3. 50 mg/mL Gentamycin stock in H 2 O. Store aliquots at
À20
C. Working concentration 50 μg/mL.
4. 150 mM 3,5-dimethoxy-4-hydroxyacetophenone (Acetosyringone) stock in DMSO. Store aliquots at À20
C.
5. 10 mg/ml (36.7 mM) β-Estradiol stock in EtOH. Store aliquots at À20
C. Working concentration 2.5–10 μg/mL.
3 Methods
3.1 Transformation
and Culture
of Agrobacterium
1. Prepare for each transformation one 1.5-mL tube.
2. Thaw for each transformation one 100 μL aliquot of Agrobacterium competent cells (stored at À80
C) on ice.
3. Add 1 μg of plasmid DNA to each tube and mix by tapping
with your index finger and keep them on ice for 3 min.
4. Transfer the tubes to liquid nitrogen to completely freeze the
samples for 3 min
5. Transfer the tubes to 37
C water bath and incubate for 3 min.
6. Immediately after the above free-thaw transformation, add
1 mL of LB medium to each 1.5-mL microcentrifuge tube.
7. Gently rotate the tubes for 4 h at 28
C (or room temperature)
using rotating mixer.
8. Spin the tubes at 1500 Â g for 5 min to get pellet cells.
9. Remove supernatant and plate 100 μL of the suspension on
fresh LB agar plates containing appropriate antibiotics. Incubate the plate for 2–3 days at 28
C until several big colonies
become visible.
10. Pick one colony from each plate and re-streak on a new LB agar
plate containing appropriate antibiotics. Incubate the plate
overnight at 28
C and keep it for the future use.
Tripartite Split-sfGFP Assay
329