1. Incubate 4 μL (2 μg) of pBS-SL24 plasmid DNA with 1 μL
(20 units) of NotI, 1 μL (20 units) of SalI, 1 μL (5 units) of
Klenow enzyme, 1 μL of 10 mM dNTP, 2 μL of enzyme buffer,
and 10 μL of sterilized water at 37
C for 2 h.
2. Incubate 4 μL (2 μg) of pCAMBIA1390-35S-FT plasmid DNA
with 1 μL (20 units) of XbaI, 1 μL (5 units) of Klenow enzyme,
1 μL of 10 mM dNTP, 2 μL of enzyme buffer, and 11 μL of
sterilized water at 37
C for 2 h to create a linearized, bluntended vector.
3. Separate the DNA fragment of blunt-ended vector as well as
the DNA fragment containing SL24 (841 bp) or SL48
(1719 bp) by electrophoresis through 0.8% agarose in TAE
buffer. Purify the vector and DNA fragments from the gel
with a gel extraction kit (see Note 5).
4. Mix 3 μL of the purified SL24/SL48 fragment with 1 μL of the
purified linearized and blunt-ended vector as well as with 1 μL
(400 units) T4 DNA ligase, 1 μL T4 DNA ligase buffer, and
4 μL sterilized water, and incubate at 16
C overnight.
5. Mix the ligation mixture with 100 μL transformationcompetent E. coli cells (see Note 5) and incubate on ice for
30 min. Select colonies of transformed bacteria on kanamycincontaining LB agar plates at 37
C for overnight.
6. Use a single colony to inoculate 3 mL of kanamycin-containing
LB medium and grow the bacteria at 37
C on a shaker set to
220 rpm for 6–8 h. Purify plasmid DNA with a plasmid DNA
isolation kit (see Note 5) and verify the insertion by sequencing
or restriction enzyme digestion.
7. Incubate 100 μL transformation-competent A. tumefaciens
cells (see Note 5) with 1 μL plasmid DNA carrying the SL24tagged FT mRNA. Incubate the bacteria cells on ice for 30 min.
Select colonies of transformed A. tumefaciens bacteria on
kanamycin-containing LB agar plates at 28
C for 2 days.
3.2 Transient
Expression
The following plasmids are required for the RNA labeling experiment (see Note 1):
pCAMBIA1390-35S-FT SL24 (mobile FT mRNA with 24 repeats
of SL).
pCAMBIA1390-35S-FT (negative control of mobile FT mRNA
without SL).
pCAMBIA1390-35S-RFP SL24 (nonmobile RFP mRNA with
24 repeats of SL).
pCAMBIA1390-35S-RFP (negative control of nonmobile RFP
mRNA without SL).
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Kai-Ren Luo et al.
(20 units) of NotI, 1 μL (20 units) of SalI, 1 μL (5 units) of
Klenow enzyme, 1 μL of 10 mM dNTP, 2 μL of enzyme buffer,
and 10 μL of sterilized water at 37
C for 2 h.
2. Incubate 4 μL (2 μg) of pCAMBIA1390-35S-FT plasmid DNA
with 1 μL (20 units) of XbaI, 1 μL (5 units) of Klenow enzyme,
1 μL of 10 mM dNTP, 2 μL of enzyme buffer, and 11 μL of
sterilized water at 37
C for 2 h to create a linearized, bluntended vector.
3. Separate the DNA fragment of blunt-ended vector as well as
the DNA fragment containing SL24 (841 bp) or SL48
(1719 bp) by electrophoresis through 0.8% agarose in TAE
buffer. Purify the vector and DNA fragments from the gel
with a gel extraction kit (see Note 5).
4. Mix 3 μL of the purified SL24/SL48 fragment with 1 μL of the
purified linearized and blunt-ended vector as well as with 1 μL
(400 units) T4 DNA ligase, 1 μL T4 DNA ligase buffer, and
4 μL sterilized water, and incubate at 16
C overnight.
5. Mix the ligation mixture with 100 μL transformationcompetent E. coli cells (see Note 5) and incubate on ice for
30 min. Select colonies of transformed bacteria on kanamycincontaining LB agar plates at 37
C for overnight.
6. Use a single colony to inoculate 3 mL of kanamycin-containing
LB medium and grow the bacteria at 37
C on a shaker set to
220 rpm for 6–8 h. Purify plasmid DNA with a plasmid DNA
isolation kit (see Note 5) and verify the insertion by sequencing
or restriction enzyme digestion.
7. Incubate 100 μL transformation-competent A. tumefaciens
cells (see Note 5) with 1 μL plasmid DNA carrying the SL24tagged FT mRNA. Incubate the bacteria cells on ice for 30 min.
Select colonies of transformed A. tumefaciens bacteria on
kanamycin-containing LB agar plates at 28
C for 2 days.
3.2 Transient
Expression
The following plasmids are required for the RNA labeling experiment (see Note 1):
pCAMBIA1390-35S-FT SL24 (mobile FT mRNA with 24 repeats
of SL).
pCAMBIA1390-35S-FT (negative control of mobile FT mRNA
without SL).
pCAMBIA1390-35S-RFP SL24 (nonmobile RFP mRNA with
24 repeats of SL).
pCAMBIA1390-35S-RFP (negative control of nonmobile RFP
mRNA without SL).
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Kai-Ren Luo et al.