DNA (primers NC-xxx-3
0 flank-F/NC-xxx-3
0 flank-R), and
(6) the AmpR-ORI fragment from pAL12-Lifeact (primers
AmpR-F/ORI-R). Use NEB Tm Calculator (https://
tmcalculator.neb.com) to select annealing temperatures.
2. Determine the correct size of the generated amplicons by
electrophoresis in a 1% agarose gel containing 0.05 μL/mL
ethidium bromide.
3. Use DpnI restriction digestion according to the manufacturer’s
instructions to remove methylated template plasmids.
4. Purify all DNA amplicons with a PCR cleanup kit according to
the manufacturer’s instructions.
5. Assemble the six DNA fragments seamlessly via their overlapping ends using the NEBuilder Assembly master mix according
to the manufacturer’s recommendations. This will place the
12xcsy stem-loops between the stop codon and 3
0 UTR of
your target gene and a hygromycin B expression cassette downstream of the terminator of your target gene (see Fig. 1c). The
entire cassette for homologous recombination is flanked by
PacI sites for release from the plasmid backbone.
6. Transform 50 μL E. coli competent cells with 2 μL NEBuilder
reaction using a standard heat shock protocol, and allow cells to
recover in 1 mL LB medium without antibiotics for 1 h at
37
C.
7. Plate 100 μL transformed cells on LB agar plates containing
100 μg/mL ampicillin and incubate at 37
C overnight.
8. Identify positive E. coli clones by colony PCR with primers
NC-xxx-IF-F/NC-xxx-3
0 flank-R and inoculate 5 mL LB liquid
medium containing 100 μg/mL ampicillin with positive colonies; incubate at 37
C overnight with shaking.
9. Purify the resulting pAL15.X-12xcsySL plasmids from each
culture using a plasmid miniprep kit according to the manufacturer’s instructions.
10. Confirm correct inserts by DNA sequencing with the primers
used to amplify the knock-in cassette.
3.8 Preparation of
Electrocompetent
Conidia of N. crassa
FGSC6103
1. Supplement 100 mL Vogel’s minimal medium with 0.5 mg/
mL L-histidine in a 500 mL conical flask and inoculate with
N. crassa strain FGSC6103.
2. Incubate for 3 days at 30
C in the dark and for another
4–6 days at room temperature on the bench in daylight, until
intensively orange conidia have developed.
3. Harvest the conidia by rinsing the flask culture with 40 mL
ice-cold 1 M sorbitol and transfer the spore suspension into a
50 mL screw-cap tube.
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David Burnett et al.
0 flank-F/NC-xxx-3
0 flank-R), and
(6) the AmpR-ORI fragment from pAL12-Lifeact (primers
AmpR-F/ORI-R). Use NEB Tm Calculator (https://
tmcalculator.neb.com) to select annealing temperatures.
2. Determine the correct size of the generated amplicons by
electrophoresis in a 1% agarose gel containing 0.05 μL/mL
ethidium bromide.
3. Use DpnI restriction digestion according to the manufacturer’s
instructions to remove methylated template plasmids.
4. Purify all DNA amplicons with a PCR cleanup kit according to
the manufacturer’s instructions.
5. Assemble the six DNA fragments seamlessly via their overlapping ends using the NEBuilder Assembly master mix according
to the manufacturer’s recommendations. This will place the
12xcsy stem-loops between the stop codon and 3
0 UTR of
your target gene and a hygromycin B expression cassette downstream of the terminator of your target gene (see Fig. 1c). The
entire cassette for homologous recombination is flanked by
PacI sites for release from the plasmid backbone.
6. Transform 50 μL E. coli competent cells with 2 μL NEBuilder
reaction using a standard heat shock protocol, and allow cells to
recover in 1 mL LB medium without antibiotics for 1 h at
37
C.
7. Plate 100 μL transformed cells on LB agar plates containing
100 μg/mL ampicillin and incubate at 37
C overnight.
8. Identify positive E. coli clones by colony PCR with primers
NC-xxx-IF-F/NC-xxx-3
0 flank-R and inoculate 5 mL LB liquid
medium containing 100 μg/mL ampicillin with positive colonies; incubate at 37
C overnight with shaking.
9. Purify the resulting pAL15.X-12xcsySL plasmids from each
culture using a plasmid miniprep kit according to the manufacturer’s instructions.
10. Confirm correct inserts by DNA sequencing with the primers
used to amplify the knock-in cassette.
3.8 Preparation of
Electrocompetent
Conidia of N. crassa
FGSC6103
1. Supplement 100 mL Vogel’s minimal medium with 0.5 mg/
mL L-histidine in a 500 mL conical flask and inoculate with
N. crassa strain FGSC6103.
2. Incubate for 3 days at 30
C in the dark and for another
4–6 days at room temperature on the bench in daylight, until
intensively orange conidia have developed.
3. Harvest the conidia by rinsing the flask culture with 40 mL
ice-cold 1 M sorbitol and transfer the spore suspension into a
50 mL screw-cap tube.
172
David Burnett et al.