3.2 Preparation of
Linear DNA Template
for In Vitro
Transcription
The purpose of this part of the protocol is to linearize the recombinant DNA template in preparation for in vitro transcription.
Include both negative (PVX/ΔCP) and positive (PVX/CP) RNA
movement controls.
1. Mix 10 μL of 10Â SpeI restriction enzyme buffer with 10 μL of
10Â BSA (1 mg/mL), 10.5 μg of plasmid DNA, and 3 μL of
SpeI (10 units/μL) with sterile distilled water to make 100 μL
in total.
2. Incubate at 37
C for 3 h.
3. Add an equal volume (100 μL) of phenol/chloroform, vortex
for 30 s, and centrifuge at 16,000 Â g at 25
C for 3 min.
4. Transfer 100 μL of the upper aqueous phase to a new
0.5 mL tube.
5. Repeat steps 3 and 4.
6. Add an equal volume (100 μL) of chloroform, vortex for 30 s,
and centrifuge at 16,000 Â g at 25
C for 3 min.
7. Transfer 100 μL of the upper aqueous phase to a new
1.5 mL tube.
8. 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), mix well, incubate at À70
C for at least 1 h,
and centrifuge at 16,000 Â g at 25
C for 15–20 min.
9. 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.
10. Carefully pipette out the supernatant and dissolve the small
pellet in 40 μL sterile distilled water to make a final concentration of 0.25 μg/μL linearized plasmid DNA.
3.3 In Vitro
Transcription
(See Note 2)
This section outlines the steps for in vitro transcription of the
linearized DNA to produce high-quality RNA inoculum for plant
infection and monitoring RNA mobility.
1. Mix 10 μL of linearized PVX DNA containing the RNA of
interest (0.25 μg/μL) with 20 μL RNase-free water, 5 μL 10Â
buffer, 1 μL RNasin (40 units/μL), 5 μL 10Â NTPs (20 mM
each of ATP, CTP, UTP, 2.0 mM GTP), and 5 μL of 5 mM Cap
analog (m7GpppG). Include linearized control plasmids for
PVX/CP and PVX/ΔCP. RNAs transcribed from these plasmids will provide positive (+CP) and negative (no CP) controls, respectively.
2. Incubate at 37
C for 5 min.
3. Add 4 μL of T7 RNA polymerase (50 units/μL) to the mixture.
4. Incubate at 37
C for 25 min.
PVX-Based RNA Movement Assay
189
Linear DNA Template
for In Vitro
Transcription
The purpose of this part of the protocol is to linearize the recombinant DNA template in preparation for in vitro transcription.
Include both negative (PVX/ΔCP) and positive (PVX/CP) RNA
movement controls.
1. Mix 10 μL of 10Â SpeI restriction enzyme buffer with 10 μL of
10Â BSA (1 mg/mL), 10.5 μg of plasmid DNA, and 3 μL of
SpeI (10 units/μL) with sterile distilled water to make 100 μL
in total.
2. Incubate at 37
C for 3 h.
3. Add an equal volume (100 μL) of phenol/chloroform, vortex
for 30 s, and centrifuge at 16,000 Â g at 25
C for 3 min.
4. Transfer 100 μL of the upper aqueous phase to a new
0.5 mL tube.
5. Repeat steps 3 and 4.
6. Add an equal volume (100 μL) of chloroform, vortex for 30 s,
and centrifuge at 16,000 Â g at 25
C for 3 min.
7. Transfer 100 μL of the upper aqueous phase to a new
1.5 mL tube.
8. 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), mix well, incubate at À70
C for at least 1 h,
and centrifuge at 16,000 Â g at 25
C for 15–20 min.
9. 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.
10. Carefully pipette out the supernatant and dissolve the small
pellet in 40 μL sterile distilled water to make a final concentration of 0.25 μg/μL linearized plasmid DNA.
3.3 In Vitro
Transcription
(See Note 2)
This section outlines the steps for in vitro transcription of the
linearized DNA to produce high-quality RNA inoculum for plant
infection and monitoring RNA mobility.
1. Mix 10 μL of linearized PVX DNA containing the RNA of
interest (0.25 μg/μL) with 20 μL RNase-free water, 5 μL 10Â
buffer, 1 μL RNasin (40 units/μL), 5 μL 10Â NTPs (20 mM
each of ATP, CTP, UTP, 2.0 mM GTP), and 5 μL of 5 mM Cap
analog (m7GpppG). Include linearized control plasmids for
PVX/CP and PVX/ΔCP. RNAs transcribed from these plasmids will provide positive (+CP) and negative (no CP) controls, respectively.
2. Incubate at 37
C for 5 min.
3. Add 4 μL of T7 RNA polymerase (50 units/μL) to the mixture.
4. Incubate at 37
C for 25 min.
PVX-Based RNA Movement Assay
189