4. MES buffer: 0.1 M MES monohydrate, pH 5.7, stored at 4
C
after sterilization.
5. LB medium: 10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast
extract, supplemented with 50 μg/ml kanamycin.
6. Acetosyringone stock solution: 0.1 M Acetosyringone in ethanol. Store at À20
C.
7. Inoculation medium: LB medium supplemented with 10 mM
MES buffer and 20 μM acetosyringone (see Note 2).
8. Incubator with shaker set to 28
C and 220 rpm.
9. Infiltration medium: 10 mM MES, 10 mM MgCl 2 , and
200 μM acetosyringone in distilled water (see Note 2).
10. 125 mL Glass flask.
11. Glass test tubes (12 Â 100 mm).
12. 15 and 50 mL Falcon polypropylene conical tubes.
13. Disposable 5 mL syringe without needle.
2.3 Imaging
1. Ophthalmic scissors (10 cm).
2. Blunt-headed forceps.
3. Pipettes and compatible tips.
4. Glass slides (76 Â 26 mm).
5. Cover slides (18 Â 18 mm, 175 Æ 5 μm thick) (see Note 3).
6. Sterilized distilled water.
7. Kimwipes (Kimberly Clark, Irving, TX).
8. Confocal laser scanning microscope (e.g., LSM880 by Carl
Zeiss, Oberkochen, Germany) equipped with 20Â, 40Â, and
63Â magnification objectives.
9. Computer with software for image acquisition (e.g., the ZEN
software developed by Carl Zeiss for LSM880 confocal laser
scanning microscope).
3 Methods
3.1 Molecular
Cloning
To track intracellular movement of mRNA, it is necessary to generate the plasmid containing the cDNA of the target mRNA fused
with multiple repeats of the MS2 stem-loop (SL) structure. Here,
we describe how to construct the plasmid encoding SL-tagged FT.
We provide two pBluescript-based plasmids, pBS-SL24 (with
24 MS2 stem-loops) and pBS-SL48 (with 48 MS2 stem-loops),
for restriction enzyme-based cloning of SL-tagged target mRNA
(Fig. 2, see Notes 1 and 4).
Plant mRNA Live-Cell Imaging Using MS2
149
C
after sterilization.
5. LB medium: 10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast
extract, supplemented with 50 μg/ml kanamycin.
6. Acetosyringone stock solution: 0.1 M Acetosyringone in ethanol. Store at À20
C.
7. Inoculation medium: LB medium supplemented with 10 mM
MES buffer and 20 μM acetosyringone (see Note 2).
8. Incubator with shaker set to 28
C and 220 rpm.
9. Infiltration medium: 10 mM MES, 10 mM MgCl 2 , and
200 μM acetosyringone in distilled water (see Note 2).
10. 125 mL Glass flask.
11. Glass test tubes (12 Â 100 mm).
12. 15 and 50 mL Falcon polypropylene conical tubes.
13. Disposable 5 mL syringe without needle.
2.3 Imaging
1. Ophthalmic scissors (10 cm).
2. Blunt-headed forceps.
3. Pipettes and compatible tips.
4. Glass slides (76 Â 26 mm).
5. Cover slides (18 Â 18 mm, 175 Æ 5 μm thick) (see Note 3).
6. Sterilized distilled water.
7. Kimwipes (Kimberly Clark, Irving, TX).
8. Confocal laser scanning microscope (e.g., LSM880 by Carl
Zeiss, Oberkochen, Germany) equipped with 20Â, 40Â, and
63Â magnification objectives.
9. Computer with software for image acquisition (e.g., the ZEN
software developed by Carl Zeiss for LSM880 confocal laser
scanning microscope).
3 Methods
3.1 Molecular
Cloning
To track intracellular movement of mRNA, it is necessary to generate the plasmid containing the cDNA of the target mRNA fused
with multiple repeats of the MS2 stem-loop (SL) structure. Here,
we describe how to construct the plasmid encoding SL-tagged FT.
We provide two pBluescript-based plasmids, pBS-SL24 (with
24 MS2 stem-loops) and pBS-SL48 (with 48 MS2 stem-loops),
for restriction enzyme-based cloning of SL-tagged target mRNA
(Fig. 2, see Notes 1 and 4).
Plant mRNA Live-Cell Imaging Using MS2
149