2.2 Agroinfiltration
2.2.1 Agrobacterium
Transformation/Growth
1. A. tumefaciens strains: C58C1 or GV3101.
2. 20 mM CaCl 2 .
3. YEB medium: 5 g/L Tryptone, 1 g/L yeast extract, 5 g/L
nutrient broth, 5 g/L sucrose 0.49 g/L MgSO4·7H 2 O.
4. YEB-RT agar plates: YEB supplemented with 1.5% (w/v) agar,
20 μg/mL rifampicin, and 5 μg/mL tetracycline.
5. YEB-RTK agar plates: YEB supplemented with 1.5% (w/v)
agar, 20 μg/mL rifampicin, 5 μg/mL tetracycline, and
50 μg/mL kanamycin.
6. YEB-RTS agar plates: YEB supplemented with 1.5% (w/v)
agar, 20 μg/mL rifampicin, 5 μg/mL tetracycline, and
100 μg/mL spectinomycin.
7. Liquid N 2 .
8. Heatable water bath.
9. Shaking incubator set to 28
C and 180–200 rpm.
10. Non-shaking incubator set to 28
C.
11. Centrifuge
2.2.2 Agroinfiltration
1. YEB medium: 5 g/L Tryptone, 1 g/L yeast extract, 5 g/L
nutrient broth, 5 g/L sucrose, 0.49 g/L MgSO4·7H 2 O.
2. Acetosyringone stock solution: 1 M Acetosyringone dissolved
in DMSO.
3. 1 M 2-(N-morpholino)ethanesulfonic acid (MES-K) (pH 5.7).
4. 1 M MgCL 2 .
5. Infiltration medium: 10 mM MgCl 2 , 250 μM acetosyringone
(prepare freshly each time from stock solutions).
6. 1-mL Hypodermic syringe.
2.3 Western Blotting
2.3.1 Protein Lysate
Preparation
1. Liquid N 2 .
2. 100 mM Phenylmethylsulfonyl fluoride (PMSF) dissolved in
isopropanol.
3. Complete protease inhibitor cocktail.
4. 200 mM Activated Na 3 VO 4 (see Note 3).
5. 1 M Dithiothreitol (DTT).
6. Lysis buffer: 10 mM Tris–HCl pH 7.6, 1 mM EDTA pH 8.0,
150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40,
5 mM NaF, 1 mM DTT, 0.5 mM Na 3 VO 4 , 1 mM PMSF,
complete protease inhibitor cocktail (the last four components
are added to the lysis buffer just before use).
7. Porcelain mortar and pestle.
8. Refrigerable microcentrifuge.
An In Vivo Reporter System for RNA Silencing
435
2.2.1 Agrobacterium
Transformation/Growth
1. A. tumefaciens strains: C58C1 or GV3101.
2. 20 mM CaCl 2 .
3. YEB medium: 5 g/L Tryptone, 1 g/L yeast extract, 5 g/L
nutrient broth, 5 g/L sucrose 0.49 g/L MgSO4·7H 2 O.
4. YEB-RT agar plates: YEB supplemented with 1.5% (w/v) agar,
20 μg/mL rifampicin, and 5 μg/mL tetracycline.
5. YEB-RTK agar plates: YEB supplemented with 1.5% (w/v)
agar, 20 μg/mL rifampicin, 5 μg/mL tetracycline, and
50 μg/mL kanamycin.
6. YEB-RTS agar plates: YEB supplemented with 1.5% (w/v)
agar, 20 μg/mL rifampicin, 5 μg/mL tetracycline, and
100 μg/mL spectinomycin.
7. Liquid N 2 .
8. Heatable water bath.
9. Shaking incubator set to 28
C and 180–200 rpm.
10. Non-shaking incubator set to 28
C.
11. Centrifuge
2.2.2 Agroinfiltration
1. YEB medium: 5 g/L Tryptone, 1 g/L yeast extract, 5 g/L
nutrient broth, 5 g/L sucrose, 0.49 g/L MgSO4·7H 2 O.
2. Acetosyringone stock solution: 1 M Acetosyringone dissolved
in DMSO.
3. 1 M 2-(N-morpholino)ethanesulfonic acid (MES-K) (pH 5.7).
4. 1 M MgCL 2 .
5. Infiltration medium: 10 mM MgCl 2 , 250 μM acetosyringone
(prepare freshly each time from stock solutions).
6. 1-mL Hypodermic syringe.
2.3 Western Blotting
2.3.1 Protein Lysate
Preparation
1. Liquid N 2 .
2. 100 mM Phenylmethylsulfonyl fluoride (PMSF) dissolved in
isopropanol.
3. Complete protease inhibitor cocktail.
4. 200 mM Activated Na 3 VO 4 (see Note 3).
5. 1 M Dithiothreitol (DTT).
6. Lysis buffer: 10 mM Tris–HCl pH 7.6, 1 mM EDTA pH 8.0,
150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40,
5 mM NaF, 1 mM DTT, 0.5 mM Na 3 VO 4 , 1 mM PMSF,
complete protease inhibitor cocktail (the last four components
are added to the lysis buffer just before use).
7. Porcelain mortar and pestle.
8. Refrigerable microcentrifuge.
An In Vivo Reporter System for RNA Silencing
435