4. T4 DNA ligase.
5. Electroporator and electroporation cuvettes with 0.2 cm electrode gap width.
6. E. coli XL1 Blue MRF’ electrocompetent cells.
7. LB liquid medium (see Subheading 2.1, item 11).
8. LB liquid medium (see Subheading 2.1, item 11) containing
100 μg/mL ampicillin.
9. LB agar plates (see Subheading 2.1, item 12) containing
100 μg/mL ampicillin.
10. Calf intestinal phosphatase (CIP).
11. For agarose gel electrophoresis:
l
Agarose.
l
TBE buffer: 89 mM Tris, 89 mM boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA).
l
Ethidium bromide.
l
Sample loading buffer (6Â): 60% glycerol, 0.3 mg/mL
bromophenol blue, 0.03 mg/mL xylene cyanol in 20 mM
Tris–HCl pH 8.
l
DNA molecular weight ladder.
l
Agarose gel tank.
l
Power supply.
l
UV transilluminator.
12. 37
C Incubator.
13. 37
C Shaking incubator.
14. Plasmid miniprep kit.
15. PCR clean up kit.
16. DNA gel extraction kit.
17. Primer PVX5539F (5
0 -TGTGTCATCAAGATTACTGG-3
0 ).
2.3 Inoculation of N.
benthamiana with
Tagged PVX by
Microprojectile
Bombardment
1. N. benthamiana plants and growth facilities suitable for
handling of transgenic plants and genetically modified plant
pathogens under containment conditions.
2. Plasmid pTRA.PVX.2xcsy.mCherry-2A-CP (see Subheading
3.2).
3. 1 mg/mL Gold particles (1 μm, BioRad 1652263) in ethanol.
4. Free-standing [18] or handheld (BioRad Helios™ 1652432)
microprojectile gun allowing microprojectile bombardment of
leaves without removing them from plants. Descriptions in
Subheading 3.3 are based on the former, which uses Swinnex
13 mm syringe filter holders (Sigma-Aldrich SX0001300) as
sample holders.
RNA Imaging with Csy4
161
5. Electroporator and electroporation cuvettes with 0.2 cm electrode gap width.
6. E. coli XL1 Blue MRF’ electrocompetent cells.
7. LB liquid medium (see Subheading 2.1, item 11).
8. LB liquid medium (see Subheading 2.1, item 11) containing
100 μg/mL ampicillin.
9. LB agar plates (see Subheading 2.1, item 12) containing
100 μg/mL ampicillin.
10. Calf intestinal phosphatase (CIP).
11. For agarose gel electrophoresis:
l
Agarose.
l
TBE buffer: 89 mM Tris, 89 mM boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA).
l
Ethidium bromide.
l
Sample loading buffer (6Â): 60% glycerol, 0.3 mg/mL
bromophenol blue, 0.03 mg/mL xylene cyanol in 20 mM
Tris–HCl pH 8.
l
DNA molecular weight ladder.
l
Agarose gel tank.
l
Power supply.
l
UV transilluminator.
12. 37
C Incubator.
13. 37
C Shaking incubator.
14. Plasmid miniprep kit.
15. PCR clean up kit.
16. DNA gel extraction kit.
17. Primer PVX5539F (5
0 -TGTGTCATCAAGATTACTGG-3
0 ).
2.3 Inoculation of N.
benthamiana with
Tagged PVX by
Microprojectile
Bombardment
1. N. benthamiana plants and growth facilities suitable for
handling of transgenic plants and genetically modified plant
pathogens under containment conditions.
2. Plasmid pTRA.PVX.2xcsy.mCherry-2A-CP (see Subheading
3.2).
3. 1 mg/mL Gold particles (1 μm, BioRad 1652263) in ethanol.
4. Free-standing [18] or handheld (BioRad Helios™ 1652432)
microprojectile gun allowing microprojectile bombardment of
leaves without removing them from plants. Descriptions in
Subheading 3.3 are based on the former, which uses Swinnex
13 mm syringe filter holders (Sigma-Aldrich SX0001300) as
sample holders.
RNA Imaging with Csy4
161