12. Transform 5 μL of the ligation in 100 μL competent E. coli
DH5α cells via the heat-shock method, plate 100 μL on LB
medium supplemented with 100 mg/L spectinomycin, and
grow overnight at 37
C.
13. Verify successful transformation via colony PCR, inoculate a
single colony in 5 mL LB medium supplemented with the
100 mg/L spectinomycin, and grow overnight at 37
C.
14. Isolate the plasmid DNA and verify successful cloning via
sequencing. Adjust the DNA concentration to 100 ng/μL.
15. The vectors are ready for transformation into Agrobacterium
strain GV3101.
3.2 Plant Material
Preparation and
AgrobacteriumMediated
Transformation
1. Grow N. benthamiana seeds in a greenhouse under 16/8h light/dark conditions and 22
C temperature for 2–4 weeks.
2. Use a single colony of Agrobacterium (step 15 from Subheading 3.1) to inoculate 5 mL LB with 100 mg/L spectinomycin
and 50 mg/L rifampicin and grow overnight at 28
C.
3. Use 1 mL of the overnight culture to inoculate 25 mL LB
medium with 100 mg/L spectinomycin and 50 mg/L rifampicin and grow overnight.
4. Measure the optical density (OD600) of the overnight culture
with a spectrometer.
5. Precipitate the bacteria in 50 mL tubes by centrifugation at
5000 Â g for 15 min.
6. Meanwhile, prepare the resuspension solution by adding
100 μM acetosyringone. Acetosyringone should always be
added to the resuspension solution after autoclaving and before
injection of leaves.
7. Resuspend the bacterial pellet in resuspension solution and
adjust the final A 600 to 0.4.
8. Leave the bacterial suspension solution on the bench at room
temperature for 2–3 h (or overnight) before infiltration.
9. Perform the infiltration with syringe. Simply press the syringe
(without needle) on the abaxial surface of the leaf and exert a
counter-pressure with finger on the other side (Fig. 4). Press
the plunger of the syringe and allow the liquid to enter the leaf
tissue. Successful infiltration is often observed as a spreading
“wetting” area in the leaf.
10. Keep the injected plants in the greenhouse at 16/8-h light/
dark conditions and 22
C temperature for 48 h.
11. Look for the occurrence of fluorescent signals with a microscope 2–4 days after injection (Fig. 5) (see Note 12).
350
Solmaz Khosravi et al.
DH5α cells via the heat-shock method, plate 100 μL on LB
medium supplemented with 100 mg/L spectinomycin, and
grow overnight at 37
C.
13. Verify successful transformation via colony PCR, inoculate a
single colony in 5 mL LB medium supplemented with the
100 mg/L spectinomycin, and grow overnight at 37
C.
14. Isolate the plasmid DNA and verify successful cloning via
sequencing. Adjust the DNA concentration to 100 ng/μL.
15. The vectors are ready for transformation into Agrobacterium
strain GV3101.
3.2 Plant Material
Preparation and
AgrobacteriumMediated
Transformation
1. Grow N. benthamiana seeds in a greenhouse under 16/8h light/dark conditions and 22
C temperature for 2–4 weeks.
2. Use a single colony of Agrobacterium (step 15 from Subheading 3.1) to inoculate 5 mL LB with 100 mg/L spectinomycin
and 50 mg/L rifampicin and grow overnight at 28
C.
3. Use 1 mL of the overnight culture to inoculate 25 mL LB
medium with 100 mg/L spectinomycin and 50 mg/L rifampicin and grow overnight.
4. Measure the optical density (OD600) of the overnight culture
with a spectrometer.
5. Precipitate the bacteria in 50 mL tubes by centrifugation at
5000 Â g for 15 min.
6. Meanwhile, prepare the resuspension solution by adding
100 μM acetosyringone. Acetosyringone should always be
added to the resuspension solution after autoclaving and before
injection of leaves.
7. Resuspend the bacterial pellet in resuspension solution and
adjust the final A 600 to 0.4.
8. Leave the bacterial suspension solution on the bench at room
temperature for 2–3 h (or overnight) before infiltration.
9. Perform the infiltration with syringe. Simply press the syringe
(without needle) on the abaxial surface of the leaf and exert a
counter-pressure with finger on the other side (Fig. 4). Press
the plunger of the syringe and allow the liquid to enter the leaf
tissue. Successful infiltration is often observed as a spreading
“wetting” area in the leaf.
10. Keep the injected plants in the greenhouse at 16/8-h light/
dark conditions and 22
C temperature for 48 h.
11. Look for the occurrence of fluorescent signals with a microscope 2–4 days after injection (Fig. 5) (see Note 12).
350
Solmaz Khosravi et al.