10. DpnI restriction enzyme and 10Â buffer.
11. PCR cleanup kit.
12. NEBuilder HiFi DNA Assembly Master Mix (NEB E2621S).
13. E. coli chemically competent cells (any standard cloning strain
suitable).
14. LB liquid medium (see Subheading 2.1, item 11).
15. LB liquid medium (see Subheading 2.1, item 11) containing
100 μg/mL ampicillin.
16. LB agar plates (see Subheading 2.1, item 12) containing
100 μg/mL ampicillin.
17. 37
C Incubator.
18. 37
C Shaking incubator.
19. Plasmid miniprep kit.
2.8 Preparation of
Electrocompetent
Conidia of N. crassa
FGSC6103
1. N. crassa strain FGSC6103 (see Note 4).
2. 1 M Sorbitol in distilled water, sterile filtered.
3. Vogel’s minimal medium [24].
4. 25 mg/mL L-Histidine in distilled water, sterile filtered.
5. 500 mL Conical flask, sterilized by autoclaving.
6. 30
C Shaking incubator.
7. Miracloth, autoclaved.
8. Funnel, autoclaved.
9. 50 mL Screw-cap tubes, sterile.
10. Refrigerated benchtop centrifuge suitable for 50 mL screw-cap
tubes.
11. Vortex mixer.
2.9 ElectroTransformation of
Csy4∗ Expression
Plasmid into N. crassa
1. pAL13.Csy4∗-GFP plasmid (see Subheading 3.6).
2. SspI and PciI restriction enzymes with corresponding 10Â
buffers.
3. PCR cleanup kit.
4. N. crassa strain FGSC6103 electrocompetent conidia (see Subheading 3.8).
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 dimethyl sulfoxide (DMSO).
9. 15 mL Screw-cap tubes, sterile.
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