10. Vogel’s minimal medium top agar (prepared using Vogel’s
minimal liquid medium as per Subheading 2.4, item 3, with
the addition of 7 g/L agar), molten, and kept liquid at 55
C.
11. Vogel’s minimal medium bottom (thinly poured) agar plates
(prepared using Vogel’s minimal liquid medium as per Subheading 2.4, item 3, with the addition of 15 g/L agar).
12. Vogel’s minimal medium agar plates (prepared using Vogel’s
minimal liquid medium as per Subheading 2.4, item 3, with
the addition of 15 g/L agar).
13. 30
C Incubator.
14. 30
C Shaking incubator.
2.10 ElectroTransformation of the
12xcsy Stem-Loop
Knock-In Cassette into
N. crassa
1. Plasmid pAL15.X-12xcsySL containing 12xcsy stem-loop
knock-in cassette (see Subheading 3.7).
2. PacI restriction enzyme with corresponding 10Â buffer.
3. PCR cleanup kit.
4. N. crassa transformant strain expressing Csy4∗-GFP from his3
locus (see Subheading 3.9).
5. 1 M Sorbitol in distilled water, sterile filtered.
6. Electroporator and electroporation cuvettes with 0.2 cm electrode gap width.
7. Yeast extract.
8. 50 μm SCR7 pyrazine in DMSO.
9. 15 mL Screw-cap tubes, sterile.
10. Vogel’s minimal medium top agar (see Subheading 2.9,
item 10) supplemented with 50 μg/mL hygromycin B, molten, and kept liquid at 55
C.
11. Vogel’s minimal medium bottom (thinly poured) agar plates
(see Subheading 2.9, item 11) supplemented with 250 μg/mL
hygromycin B.
12. Vogel’s minimal medium agar plates (see Subheading 2.9,
item 12) supplemented with 200 μg/mL hygromycin B.
13. 30
C Incubator.
14. 30
C Shaking incubator.
2.11 Visualization of
Tagged mRNA in
N. crassa
1. Inverted confocal laser scanning microscope equipped with
40Â and 63Â oil immersion lenses, capable of imaging GFP.
2. Colony of N. crassa transformant strain expressing Csy4∗-GFP
from his3 locus (see Subheading 3.9) and 12xcsy-tagged mRNA
(see Subheading 3.10) cultivated on Vogel’s minimal medium
agar plates (see Subheading 2.9, item 9) supplemented with
200 μg/mL hygromycin B.
RNA Imaging with Csy4
165
minimal liquid medium as per Subheading 2.4, item 3, with
the addition of 7 g/L agar), molten, and kept liquid at 55
C.
11. Vogel’s minimal medium bottom (thinly poured) agar plates
(prepared using Vogel’s minimal liquid medium as per Subheading 2.4, item 3, with the addition of 15 g/L agar).
12. Vogel’s minimal medium agar plates (prepared using Vogel’s
minimal liquid medium as per Subheading 2.4, item 3, with
the addition of 15 g/L agar).
13. 30
C Incubator.
14. 30
C Shaking incubator.
2.10 ElectroTransformation of the
12xcsy Stem-Loop
Knock-In Cassette into
N. crassa
1. Plasmid pAL15.X-12xcsySL containing 12xcsy stem-loop
knock-in cassette (see Subheading 3.7).
2. PacI restriction enzyme with corresponding 10Â buffer.
3. PCR cleanup kit.
4. N. crassa transformant strain expressing Csy4∗-GFP from his3
locus (see Subheading 3.9).
5. 1 M Sorbitol in distilled water, sterile filtered.
6. Electroporator and electroporation cuvettes with 0.2 cm electrode gap width.
7. Yeast extract.
8. 50 μm SCR7 pyrazine in DMSO.
9. 15 mL Screw-cap tubes, sterile.
10. Vogel’s minimal medium top agar (see Subheading 2.9,
item 10) supplemented with 50 μg/mL hygromycin B, molten, and kept liquid at 55
C.
11. Vogel’s minimal medium bottom (thinly poured) agar plates
(see Subheading 2.9, item 11) supplemented with 250 μg/mL
hygromycin B.
12. Vogel’s minimal medium agar plates (see Subheading 2.9,
item 12) supplemented with 200 μg/mL hygromycin B.
13. 30
C Incubator.
14. 30
C Shaking incubator.
2.11 Visualization of
Tagged mRNA in
N. crassa
1. Inverted confocal laser scanning microscope equipped with
40Â and 63Â oil immersion lenses, capable of imaging GFP.
2. Colony of N. crassa transformant strain expressing Csy4∗-GFP
from his3 locus (see Subheading 3.9) and 12xcsy-tagged mRNA
(see Subheading 3.10) cultivated on Vogel’s minimal medium
agar plates (see Subheading 2.9, item 9) supplemented with
200 μg/mL hygromycin B.
RNA Imaging with Csy4
165