a thermocycler at 22
C for 5 min followed by 42
C for 30 min
and a final step at 85
C for 5 min. Store the newly formed
cDNA at À20
C.
12. For the production of a DNA fragment flanked by T7 promoter sequences perform a simple PCR with the T7 primers
(the T7 sequence 5
0 -TAATACGACTCACTATAGGG-3
0 must
be attached at the 5
0 ends of each primer to permit the association of T7 polymerase with the DNA fragment and the latter
production of labeled dsRNA). As the template for the PCR
with these primers use the cDNA previously produced. In a
PCR collection tube mix around 500 ng of genomic templates
with 0.1–0.5 μM of T7-primers, 1.5 mM of MgCl 2 , 0.5 U of
Taq polymerase plus buffer, 4 mM deoxynucleotides (dNTPs),
and RNase-free water to reach a total volume of 25 μL (see
Note 6).
13. Set up cycler parameters (annealing temperature, denaturation,
and extension time) according to your primers, length of the
amplicon, and Taq polymerase (see Note 7).
14. After completion of the PCR reaction, run the total PCR
amplicon on an agarose gel (1.2%) to verify the correct length
of the DNA fragment.
15. Extract the DNA fragment from the gel with the help of a
scalpel, and place it in a 2 mL centrifuge micro-collection tube.
16. For purifying the DNA fragment use any PCR cleanup kit: add
10 μL of membrane-binding solution per 10 mg of gel slice,
vortex briefly, and incubate at 55
C until the gel is completely
dissolved.
17. Place a silica membrane spin column into a collection tube,
transfer the dissolved gel mixture into it, incubate for 1 min,
centrifuge at 16,000 Â g at RT for 1 min, and discard the flowthrough.
18. Perform two washing steps with membrane wash solution (the
first one with 700 μL and the second one with 500 μL) each
followed by centrifugation (16,000 Â g at RT for 2 min).
Discard again the flow-through, and dry the membrane with
a final centrifugation (16,000 Â g at RT for 1 min).
19. Finally, add 50 μL of nuclease-free water to the silica membrane spin column and centrifuge for a last time (16,000 Â g at
RT for 1 min). Keep the purified DNA at 4
C for a short time
or store it at À20
C.
20. For labeling of dsRNA: Apply approximately 1 μg of linearized
T7 DNA template for bidirectional in vitro transcription (see
Note 8) in the presence of fluorescent Atto 488 UTP to create
Atto 488 -UTP-tagged dsRNA. Using components of an
Atto488 RNA Labeling Kit and following the instruction
Labeling of dsRNA for Fungal Uptake Detection Analysis
233
C for 5 min followed by 42
C for 30 min
and a final step at 85
C for 5 min. Store the newly formed
cDNA at À20
C.
12. For the production of a DNA fragment flanked by T7 promoter sequences perform a simple PCR with the T7 primers
(the T7 sequence 5
0 -TAATACGACTCACTATAGGG-3
0 must
be attached at the 5
0 ends of each primer to permit the association of T7 polymerase with the DNA fragment and the latter
production of labeled dsRNA). As the template for the PCR
with these primers use the cDNA previously produced. In a
PCR collection tube mix around 500 ng of genomic templates
with 0.1–0.5 μM of T7-primers, 1.5 mM of MgCl 2 , 0.5 U of
Taq polymerase plus buffer, 4 mM deoxynucleotides (dNTPs),
and RNase-free water to reach a total volume of 25 μL (see
Note 6).
13. Set up cycler parameters (annealing temperature, denaturation,
and extension time) according to your primers, length of the
amplicon, and Taq polymerase (see Note 7).
14. After completion of the PCR reaction, run the total PCR
amplicon on an agarose gel (1.2%) to verify the correct length
of the DNA fragment.
15. Extract the DNA fragment from the gel with the help of a
scalpel, and place it in a 2 mL centrifuge micro-collection tube.
16. For purifying the DNA fragment use any PCR cleanup kit: add
10 μL of membrane-binding solution per 10 mg of gel slice,
vortex briefly, and incubate at 55
C until the gel is completely
dissolved.
17. Place a silica membrane spin column into a collection tube,
transfer the dissolved gel mixture into it, incubate for 1 min,
centrifuge at 16,000 Â g at RT for 1 min, and discard the flowthrough.
18. Perform two washing steps with membrane wash solution (the
first one with 700 μL and the second one with 500 μL) each
followed by centrifugation (16,000 Â g at RT for 2 min).
Discard again the flow-through, and dry the membrane with
a final centrifugation (16,000 Â g at RT for 1 min).
19. Finally, add 50 μL of nuclease-free water to the silica membrane spin column and centrifuge for a last time (16,000 Â g at
RT for 1 min). Keep the purified DNA at 4
C for a short time
or store it at À20
C.
20. For labeling of dsRNA: Apply approximately 1 μg of linearized
T7 DNA template for bidirectional in vitro transcription (see
Note 8) in the presence of fluorescent Atto 488 UTP to create
Atto 488 -UTP-tagged dsRNA. Using components of an
Atto488 RNA Labeling Kit and following the instruction
Labeling of dsRNA for Fungal Uptake Detection Analysis
233