2 Materials
2.1 Inducible Vector
Cloning
1. Gel-purified PCR-amplified products of gene of interest and
BirA* (see Note 2).
2. An inducible vector system like pDEX BASTA (pDB) vector
carrying dexamethasone (DEX)-inducible GVG gene (see
Notes 3 and 4; Fig. 1a).
3. StuI restriction enzyme.
4. Gel purification kit.
5. LB plates with Kanamycin (Kan) selection.
6. E. coli DH5α competent cells.
7. Liquid LB with Kan selection.
8. Primers for colony PCR.
9. Competent Agrobacterium tumefaciens GV3101 cells.
10. LB plates with Rifampicin (Rif) and Kan selection.
2.2 Plant
Transformation
1. Four-week-old Arabidopsis thaliana (ecotype Col-0) plants
with 5–10 cm bolts (3 plants per construct; grown in
medium-sized pots in 16-h light at 22
C).
2. Liquid LB with Rif and Kan selection.
3. Arabidopsis infiltration media: 10 mM MgCl 2 , 50 g/L
Sucrose, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid
(MES). Bring the solution to pH 5.6 with 5 M potassium
hydroxide.
4. Silwet L-77.
5. 300 mL Beaker.
6. Plastic wrap.
7. Spray bottle with water.
8. Large centrifuge at 4
C.
9. Autoclaved 250 mL culture bottles for centrifugation.
10. Dark tray or cloth.
2.3 Transgenic
Arabidopsis Selection
and Expression
1. Basta (60 g/L glufosinate ammonium).
2. Half of a 1.5 mL tube full of transformed seeds
(T0 generation).
3. Spray bottle.
4. Fume hood.
5. Large tray of soil.
6. HA antibody.
7. Goat anti-Rat IgG-HRP secondary antibody.
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Madiha Khan et al.
2.1 Inducible Vector
Cloning
1. Gel-purified PCR-amplified products of gene of interest and
BirA* (see Note 2).
2. An inducible vector system like pDEX BASTA (pDB) vector
carrying dexamethasone (DEX)-inducible GVG gene (see
Notes 3 and 4; Fig. 1a).
3. StuI restriction enzyme.
4. Gel purification kit.
5. LB plates with Kanamycin (Kan) selection.
6. E. coli DH5α competent cells.
7. Liquid LB with Kan selection.
8. Primers for colony PCR.
9. Competent Agrobacterium tumefaciens GV3101 cells.
10. LB plates with Rifampicin (Rif) and Kan selection.
2.2 Plant
Transformation
1. Four-week-old Arabidopsis thaliana (ecotype Col-0) plants
with 5–10 cm bolts (3 plants per construct; grown in
medium-sized pots in 16-h light at 22
C).
2. Liquid LB with Rif and Kan selection.
3. Arabidopsis infiltration media: 10 mM MgCl 2 , 50 g/L
Sucrose, 0.5 g/L 2-(N-morpholino) ethanesulfonic acid
(MES). Bring the solution to pH 5.6 with 5 M potassium
hydroxide.
4. Silwet L-77.
5. 300 mL Beaker.
6. Plastic wrap.
7. Spray bottle with water.
8. Large centrifuge at 4
C.
9. Autoclaved 250 mL culture bottles for centrifugation.
10. Dark tray or cloth.
2.3 Transgenic
Arabidopsis Selection
and Expression
1. Basta (60 g/L glufosinate ammonium).
2. Half of a 1.5 mL tube full of transformed seeds
(T0 generation).
3. Spray bottle.
4. Fume hood.
5. Large tray of soil.
6. HA antibody.
7. Goat anti-Rat IgG-HRP secondary antibody.
430
Madiha Khan et al.