Reagent
Volume (μL)
Template DNA, final amount recommended should be
between 50 and 250 ng if genomic, 1–10 ng if using a
plasmid template
x
5Â HF buffer
10
10 μM Forward primer
2.5
10 μM Reverse primer
2.5
2.5 μM dNTP
4
Phusion high-fidelity DNA polymerase
0.5
Add nuclease-free water to a final volume of 50 μL
x
2. Place in a thermocycler and run the following program, optimized for specific target DNA:
1. 98
C for 1 min
2. 98
C for 10 s
3. 45–72
C for 3 s
4. 72
C for 15–30 s (depends on the length of PCR product, 30 s per
1 kb for Phusion polymerase)
5. Repeat steps 2–4 35Â
6. 72
C for 5 min
Fig. 1 Key steps in the synthesis of fluorescein-labeled dsRNA and sRNAs
Visualizing Fungal RNA Uptake
219
Volume (μL)
Template DNA, final amount recommended should be
between 50 and 250 ng if genomic, 1–10 ng if using a
plasmid template
x
5Â HF buffer
10
10 μM Forward primer
2.5
10 μM Reverse primer
2.5
2.5 μM dNTP
4
Phusion high-fidelity DNA polymerase
0.5
Add nuclease-free water to a final volume of 50 μL
x
2. Place in a thermocycler and run the following program, optimized for specific target DNA:
1. 98
C for 1 min
2. 98
C for 10 s
3. 45–72
C for 3 s
4. 72
C for 15–30 s (depends on the length of PCR product, 30 s per
1 kb for Phusion polymerase)
5. Repeat steps 2–4 35Â
6. 72
C for 5 min
Fig. 1 Key steps in the synthesis of fluorescein-labeled dsRNA and sRNAs
Visualizing Fungal RNA Uptake
219