3. Mix gently and centrifuge briefly for 1000 Â g.
4. Incubate at 37
C for 20 min.
5. Add 100 μL 1:1 phenol/chloroform, vortex for 30 s, and
centrifuge at 16,000 Â g at 25
C for 3 min.
6. Transfer 100 μL of the upper aqueous phase to a new
1.5 mL tube.
7. Add 100 μL chloroform, vortex for 30 s, and centrifuge at
16,000 Â g at 25
C for 3 min.
8. Transfer 100 μL of the upper aqueous phase to a new
1.5 mL tube.
9. Add 10 μL of 3 M NaAc (pH 5.2) (0.1 volume of the total
water phase) and 250 μL of 100% ethanol (2.5 volumes of the
total water phase) and mix well.
10. Centrifuge at 16,000 Â g at 25
C for 15–20 min.
11. Carefully discard the supernatant, add 100 μL of 70% ethanol
to wash the small pellet (do not pipette or vortex), and centrifuge at 16,000 Â g at 25
C for 5 min.
12. Carefully remove the supernatant with a pipette and allow the
pellet to air-dry for 5–10 min.
13. Dissolve RNA in 40 μL RNase-free water (pH 7.0). RNA is
now ready for amplification by RT-PCR.
14. For amplification, set up a 15 μL RT-PCR reaction by adding
7.5 μL of One-Step SYBR Green Master Mix and 0.3 μL of
qScript One-Step Reverse Transcriptase to 50 ng of the isolated
plant RNA and 200 nM of GOI-specific forward and reverse
primers. Use GOI primers designed to contain the DNA
sequence encoding the RNA to be tested, the appropriate
restriction site for cloning into the PVX vector (MluI and
EcoRV sites), and a gene-specific tag to distinguish leafinoculated mRNA from WT RNA. We use a histidine-tag
sequence in the reverse primer for inoculum-specific detection
(Fig. 3). Use standard PCR thermocycling conditions.
15. Clone the amplified cDNA of the ROI into the MluI and
EcoRV sites within the MCS of the PVX vector using standard
cloning methods.
16. Transform the recombinant plasmid DNA into E. coli strain
DHα. Grow the bacteria under ampicillin selection and isolate
the plasmid DNA using a miniprep kit from any commercial
source. We commonly use the Qiagen miniprep kit, but other
commercial brands are suitable.
17. Verify the plasmid by DNA sequencing.
18. Measure the concentration of the plasmid DNA with a nanodrop spectrophotometer using standard methods.
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Zhiming Yu et al.
4. Incubate at 37
C for 20 min.
5. Add 100 μL 1:1 phenol/chloroform, vortex for 30 s, and
centrifuge at 16,000 Â g at 25
C for 3 min.
6. Transfer 100 μL of the upper aqueous phase to a new
1.5 mL tube.
7. Add 100 μL chloroform, vortex for 30 s, and centrifuge at
16,000 Â g at 25
C for 3 min.
8. Transfer 100 μL of the upper aqueous phase to a new
1.5 mL tube.
9. Add 10 μL of 3 M NaAc (pH 5.2) (0.1 volume of the total
water phase) and 250 μL of 100% ethanol (2.5 volumes of the
total water phase) and mix well.
10. Centrifuge at 16,000 Â g at 25
C for 15–20 min.
11. Carefully discard the supernatant, add 100 μL of 70% ethanol
to wash the small pellet (do not pipette or vortex), and centrifuge at 16,000 Â g at 25
C for 5 min.
12. Carefully remove the supernatant with a pipette and allow the
pellet to air-dry for 5–10 min.
13. Dissolve RNA in 40 μL RNase-free water (pH 7.0). RNA is
now ready for amplification by RT-PCR.
14. For amplification, set up a 15 μL RT-PCR reaction by adding
7.5 μL of One-Step SYBR Green Master Mix and 0.3 μL of
qScript One-Step Reverse Transcriptase to 50 ng of the isolated
plant RNA and 200 nM of GOI-specific forward and reverse
primers. Use GOI primers designed to contain the DNA
sequence encoding the RNA to be tested, the appropriate
restriction site for cloning into the PVX vector (MluI and
EcoRV sites), and a gene-specific tag to distinguish leafinoculated mRNA from WT RNA. We use a histidine-tag
sequence in the reverse primer for inoculum-specific detection
(Fig. 3). Use standard PCR thermocycling conditions.
15. Clone the amplified cDNA of the ROI into the MluI and
EcoRV sites within the MCS of the PVX vector using standard
cloning methods.
16. Transform the recombinant plasmid DNA into E. coli strain
DHα. Grow the bacteria under ampicillin selection and isolate
the plasmid DNA using a miniprep kit from any commercial
source. We commonly use the Qiagen miniprep kit, but other
commercial brands are suitable.
17. Verify the plasmid by DNA sequencing.
18. Measure the concentration of the plasmid DNA with a nanodrop spectrophotometer using standard methods.
188
Zhiming Yu et al.