9. Agarose.
10. TAE buffer: 40 mM Tris, 1 mM EDTA, 20 mM acetic acid.
11. Sterilized water.
12. Commercialized E. coli or Agrobacterium tumefaciens competent cells.
13. DNA gel extraction kit.
14. DNA isolation kit.
2.2 Transient
Expression
1. Agrobacteria (Agrobacterium tumefaciens strain AGL1) transformed with (see Note 1):
(a) pCAMBIA1390-35S-MS2 FD -GFP.
(b) pCAMBIA1390-35S-FT.
(c) pCAMBIA1390-35S-RFP.
(d) pCAMBIA1390-35S-FT SL24 .
(e) pCAMBIA1390-35S-RFP SL24 .
2. One-month-old Nicotiana benthamiana plants kept at 28
C
in 14-h/10-h light/dark cycle growth chambers.
3. Pipettes and compatible tips.
Fig. 2 Maps of SL3, pBS-SL24, and pBS-SL48. SL3 refers to a DNA fragment encoding three tandem repeats
of the MS2 RNA stem-loop (SL) structure and is derived from AdMl-M3 [29]. pBS-SL24 and pBS-SL48 are
pBluescript-based plasmids that contain 8 and 16 copies of SL3, respectively. The single-cut enzymes that
are used to excise the SL24 or SL48 DNA fragments are shown
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Kai-Ren Luo et al.
10. TAE buffer: 40 mM Tris, 1 mM EDTA, 20 mM acetic acid.
11. Sterilized water.
12. Commercialized E. coli or Agrobacterium tumefaciens competent cells.
13. DNA gel extraction kit.
14. DNA isolation kit.
2.2 Transient
Expression
1. Agrobacteria (Agrobacterium tumefaciens strain AGL1) transformed with (see Note 1):
(a) pCAMBIA1390-35S-MS2 FD -GFP.
(b) pCAMBIA1390-35S-FT.
(c) pCAMBIA1390-35S-RFP.
(d) pCAMBIA1390-35S-FT SL24 .
(e) pCAMBIA1390-35S-RFP SL24 .
2. One-month-old Nicotiana benthamiana plants kept at 28
C
in 14-h/10-h light/dark cycle growth chambers.
3. Pipettes and compatible tips.
Fig. 2 Maps of SL3, pBS-SL24, and pBS-SL48. SL3 refers to a DNA fragment encoding three tandem repeats
of the MS2 RNA stem-loop (SL) structure and is derived from AdMl-M3 [29]. pBS-SL24 and pBS-SL48 are
pBluescript-based plasmids that contain 8 and 16 copies of SL3, respectively. The single-cut enzymes that
are used to excise the SL24 or SL48 DNA fragments are shown
148
Kai-Ren Luo et al.