8. Pellet the germlings for 3 min at 700 Â g, remove the supernatant, and wash once with 5 mL liquid Vogel’s minimal medium.
Repeat the centrifugation and remove the supernatant.
9. Resuspend the germling pellet in 1 mL liquid Vogel’s minimal
medium, add into 30 mL liquid (55
C) Vogel’s minimal
medium top agar, and then mix by gently inverting the tube
2–3 times.
10. Distribute 5 mL top agar aliquots onto six individual
pre-warmed (30
C) Vogel’s minimal medium bottom agar
plates and wait for the top agar to solidify.
11. Incubate the plates at 30
C in the dark, and watch for
emerging colonies after 3 days.
12. Transfer emerging colonies onto fresh Vogel’s minimal
medium agar plates.
3.10 ElectroTransformation of the
12xcsy Stem-Loop
Knock-In Cassette into
N. crassa
1. Release the 12xcsy stem-loop knock-in cassette from the
pAL15.X-12xcsySL plasmid (Subheading 3.7) by PacI restriction digestion according to the manufacturer’s instructions.
2. Purify the DNA with a PCR cleanup kit according to the
manufacturer’s instructions.
3. Follow the electroporation protocol steps 3–8 as explained
above (Subheading 3.9).
4. Resuspend the germling pellet in 1 mL liquid Vogel’s minimal
medium, add to 30 mL liquid (55
C) Vogel’s minimal
medium top agar supplemented with 50 μg/mL hygromycin B,
and then mix by gently inverting the tube 2–3 times.
5. Distribute 5 mL top agar aliquots onto six individual
pre-warmed (30
C) Vogel’s minimal medium bottom agar
plates supplemented with 250 μg/mL hygromycin B and wait
for the top agar to solidify.
6. Incubate the plates at 30
C in the dark and watch for emerging
colonies after 3 days.
7. Transfer emerging colonies onto fresh Vogel’s minimal
medium agar plates supplemented with 200 μg/mL
hygromycin B.
3.11 Visualization of
Tagged mRNA in N.
crassa
1. Pre-culture a medium-size (approx. 4–5 cm diameter) fungal
colony of the final transformant strain generated in Subheading
3.10 by incubation on Vogel’s minimal medium agar supplemented with 200 μg/mL hygromycin B at 30
C for 12–14 h.
2. Use the inverted agar block method [25] to mount a
1.5 Â 1.5 cm
2 sample from the colony edge on a glass cover
slide using 15 μL of physiological salt solution as mounting
medium.
174
David Burnett et al.
Repeat the centrifugation and remove the supernatant.
9. Resuspend the germling pellet in 1 mL liquid Vogel’s minimal
medium, add into 30 mL liquid (55
C) Vogel’s minimal
medium top agar, and then mix by gently inverting the tube
2–3 times.
10. Distribute 5 mL top agar aliquots onto six individual
pre-warmed (30
C) Vogel’s minimal medium bottom agar
plates and wait for the top agar to solidify.
11. Incubate the plates at 30
C in the dark, and watch for
emerging colonies after 3 days.
12. Transfer emerging colonies onto fresh Vogel’s minimal
medium agar plates.
3.10 ElectroTransformation of the
12xcsy Stem-Loop
Knock-In Cassette into
N. crassa
1. Release the 12xcsy stem-loop knock-in cassette from the
pAL15.X-12xcsySL plasmid (Subheading 3.7) by PacI restriction digestion according to the manufacturer’s instructions.
2. Purify the DNA with a PCR cleanup kit according to the
manufacturer’s instructions.
3. Follow the electroporation protocol steps 3–8 as explained
above (Subheading 3.9).
4. Resuspend the germling pellet in 1 mL liquid Vogel’s minimal
medium, add to 30 mL liquid (55
C) Vogel’s minimal
medium top agar supplemented with 50 μg/mL hygromycin B,
and then mix by gently inverting the tube 2–3 times.
5. Distribute 5 mL top agar aliquots onto six individual
pre-warmed (30
C) Vogel’s minimal medium bottom agar
plates supplemented with 250 μg/mL hygromycin B and wait
for the top agar to solidify.
6. Incubate the plates at 30
C in the dark and watch for emerging
colonies after 3 days.
7. Transfer emerging colonies onto fresh Vogel’s minimal
medium agar plates supplemented with 200 μg/mL
hygromycin B.
3.11 Visualization of
Tagged mRNA in N.
crassa
1. Pre-culture a medium-size (approx. 4–5 cm diameter) fungal
colony of the final transformant strain generated in Subheading
3.10 by incubation on Vogel’s minimal medium agar supplemented with 200 μg/mL hygromycin B at 30
C for 12–14 h.
2. Use the inverted agar block method [25] to mount a
1.5 Â 1.5 cm
2 sample from the colony edge on a glass cover
slide using 15 μL of physiological salt solution as mounting
medium.
174
David Burnett et al.