4. Vigorously vortex the cell suspension for several minutes, and
then filter through four layers of sterile Miracloth fixed in a
sterile plastic funnel into a fresh 50 mL screw-cap tube.
5. Pellet conidia by centrifugation for 5 min at 600 Â g and 4
C,
decant supernatant, and keep cell pellet on ice.
6. Wash the conidia twice in 45 mL of ice-cold 1 M sorbitol with
centrifugation for 5 min at 800 Â g and 4
C, and finally drain
off supernatant by inverting the tube onto a paper towel.
7. Resuspend the conidia by gently vortexing the pellet in remaining runoff supernatant which should result in a viscous and
deeply orange cell suspension. If it is not possible to properly
pipette the cell suspension through a 200 μL pipette tip, dilute
with 1 M sorbitol.
8. Prepare 90 μL aliquots in pre-chilled 1.5 mL tubes and keep on
ice for immediate use.
3.9 Electro-Transformation of Csy4∗
Expression Plasmid
into N. crassa
1. Release the his3-targeted Csy4∗ expression cassette from the
pAL13.Csy4∗-GFP plasmid (Subheading 3.6) by SspI/PciI
double digestion according to the manufacturer’s instructions
(see Note 14).
2. Purify DNA with a PCR cleanup kit according to the manufacturer’s instructions.
3. Add 10 μL of transforming DNA solution (1 μg/μL) to 90 μL
of electrocompetent conidia (Subheading 3.8) and incubate for
15 min on ice.
4. Transfer the 100 μL transformation mixture into a pre-chilled
(À20
C) electroporation cuvette and remove possible air bubbles from between the electrodes by gently tapping the cuvette
on the bench surface.
5. Wipe the outside of the cuvette dry before placing it into the
shocking pod and apply one electroshock with the following
settings: exponential decay, 1.5 kV with 0.2 cm gap width
(resulting in 7.5 kV/cm field strength), 600 Ω and 25 μF
(resulting in an optimal time constant of 13.4 ms).
6. Add 900 μL of ice-cold 1 M sorbitol solution into the cuvette
immediately after the shock, and transfer the cell suspension
back into its original tube and onto ice.
7. Add 1 mL electroporated conidia to 5 mL liquid Vogel’s minimal medium supplemented with 0.01 g/mL yeast extract and
50 μM SCR7 pyrazine (see Note 15) in a 15 mL screw-cap
tube, and incubate for 3 h at 30
C with gentle shaking at
150 rpm (place the tube horizontally into a shaking incubator).
RNA Imaging with Csy4
173
then filter through four layers of sterile Miracloth fixed in a
sterile plastic funnel into a fresh 50 mL screw-cap tube.
5. Pellet conidia by centrifugation for 5 min at 600 Â g and 4
C,
decant supernatant, and keep cell pellet on ice.
6. Wash the conidia twice in 45 mL of ice-cold 1 M sorbitol with
centrifugation for 5 min at 800 Â g and 4
C, and finally drain
off supernatant by inverting the tube onto a paper towel.
7. Resuspend the conidia by gently vortexing the pellet in remaining runoff supernatant which should result in a viscous and
deeply orange cell suspension. If it is not possible to properly
pipette the cell suspension through a 200 μL pipette tip, dilute
with 1 M sorbitol.
8. Prepare 90 μL aliquots in pre-chilled 1.5 mL tubes and keep on
ice for immediate use.
3.9 Electro-Transformation of Csy4∗
Expression Plasmid
into N. crassa
1. Release the his3-targeted Csy4∗ expression cassette from the
pAL13.Csy4∗-GFP plasmid (Subheading 3.6) by SspI/PciI
double digestion according to the manufacturer’s instructions
(see Note 14).
2. Purify DNA with a PCR cleanup kit according to the manufacturer’s instructions.
3. Add 10 μL of transforming DNA solution (1 μg/μL) to 90 μL
of electrocompetent conidia (Subheading 3.8) and incubate for
15 min on ice.
4. Transfer the 100 μL transformation mixture into a pre-chilled
(À20
C) electroporation cuvette and remove possible air bubbles from between the electrodes by gently tapping the cuvette
on the bench surface.
5. Wipe the outside of the cuvette dry before placing it into the
shocking pod and apply one electroshock with the following
settings: exponential decay, 1.5 kV with 0.2 cm gap width
(resulting in 7.5 kV/cm field strength), 600 Ω and 25 μF
(resulting in an optimal time constant of 13.4 ms).
6. Add 900 μL of ice-cold 1 M sorbitol solution into the cuvette
immediately after the shock, and transfer the cell suspension
back into its original tube and onto ice.
7. Add 1 mL electroporated conidia to 5 mL liquid Vogel’s minimal medium supplemented with 0.01 g/mL yeast extract and
50 μM SCR7 pyrazine (see Note 15) in a 15 mL screw-cap
tube, and incubate for 3 h at 30
C with gentle shaking at
150 rpm (place the tube horizontally into a shaking incubator).
RNA Imaging with Csy4
173