manufacturer’s instructions, and NEB Tm Calculator (https://
tmcalculator.neb.com) to select annealing temperatures (see
Note 13).
2. Amplify a linear recipient vector backbone from pAB261 by
PCR with primer pair pAB261-lin-F/pAB261-lin-R, using
HiFi DNA polymerase as above.
3. Determine the correct size of the generated amplicons by
electrophoresis in a 1% agarose gel containing 0.05 μL/mL
ethidium bromide.
4. Use DpnI restriction digestion according to the manufacturer’s
instructions to remove methylated template plasmids.
5. Purify all DNA amplicons with a PCR cleanup kit according to
the manufacturer’s instructions.
6. Assemble the three DNA fragments seamlessly via their overlapping ends using the NEBuilder Assembly master mix
according to the manufacturer’s recommendations.
7. 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.
8. Plate 100 μL transformed cells on LB agar plates containing
100 μg/mL ampicillin and incubate at 37
C overnight.
9. Identify positive E. coli clones by colony PCR with primers
Csy4-IF-F/GFP-IF-R and inoculate 5 ml LB liquid medium
containing 100 μg/mL ampicillin with positive colonies; incubate at 37
C overnight with shaking.
10. Purify resulting pAL13.Csy4∗-GFP plasmids from each culture using a plasmid miniprep kit according to the manufacturer’s instructions.
11. Confirm correct inserts by DNA sequencing with the primers
used to amplify the Csy4∗ and GFP inserts.
3.7 Construction of
the 12xcsy Stem-Loop
Knock-In Cassette for
RNA Imaging in N.
crassa
1. Amplify by PCR with HiFi DNA polymerase according to the
manufacturer’s instructions: (1) A 5
0 genomic flanking region
for homologous recombination comprising the 1 kb sequence
upstream from the 3
0 -end (incl. stop codon) of the desired
target gene ORF from genomic DNA (primers NC-xxx-IFF/NC-xxx-IF-R), (2) the 12xcsy-stem-loop repeat from
p35S.PVX.12xcsy.mCherry-2A-CP (primers 12xcsySL-F/
12xcsySL-R), (3) the 500–650 bp terminator region of your
target gene from genomic DNA (primers NC-Txxx-IF-F/NCTxxx-IF-R), (4) the hygromycin B resistance cassette from
pLS3 (primers Pgpdh-F/Tgpdh-R), (5) a 1 kb 3
0 genomic
flanking region for homologous recombination comprising
the terminator region of the desired target gene from genomic
RNA Imaging with Csy4
171
tmcalculator.neb.com) to select annealing temperatures (see
Note 13).
2. Amplify a linear recipient vector backbone from pAB261 by
PCR with primer pair pAB261-lin-F/pAB261-lin-R, using
HiFi DNA polymerase as above.
3. Determine the correct size of the generated amplicons by
electrophoresis in a 1% agarose gel containing 0.05 μL/mL
ethidium bromide.
4. Use DpnI restriction digestion according to the manufacturer’s
instructions to remove methylated template plasmids.
5. Purify all DNA amplicons with a PCR cleanup kit according to
the manufacturer’s instructions.
6. Assemble the three DNA fragments seamlessly via their overlapping ends using the NEBuilder Assembly master mix
according to the manufacturer’s recommendations.
7. 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.
8. Plate 100 μL transformed cells on LB agar plates containing
100 μg/mL ampicillin and incubate at 37
C overnight.
9. Identify positive E. coli clones by colony PCR with primers
Csy4-IF-F/GFP-IF-R and inoculate 5 ml LB liquid medium
containing 100 μg/mL ampicillin with positive colonies; incubate at 37
C overnight with shaking.
10. Purify resulting pAL13.Csy4∗-GFP plasmids from each culture using a plasmid miniprep kit according to the manufacturer’s instructions.
11. Confirm correct inserts by DNA sequencing with the primers
used to amplify the Csy4∗ and GFP inserts.
3.7 Construction of
the 12xcsy Stem-Loop
Knock-In Cassette for
RNA Imaging in N.
crassa
1. Amplify by PCR with HiFi DNA polymerase according to the
manufacturer’s instructions: (1) A 5
0 genomic flanking region
for homologous recombination comprising the 1 kb sequence
upstream from the 3
0 -end (incl. stop codon) of the desired
target gene ORF from genomic DNA (primers NC-xxx-IFF/NC-xxx-IF-R), (2) the 12xcsy-stem-loop repeat from
p35S.PVX.12xcsy.mCherry-2A-CP (primers 12xcsySL-F/
12xcsySL-R), (3) the 500–650 bp terminator region of your
target gene from genomic DNA (primers NC-Txxx-IF-F/NCTxxx-IF-R), (4) the hygromycin B resistance cassette from
pLS3 (primers Pgpdh-F/Tgpdh-R), (5) a 1 kb 3
0 genomic
flanking region for homologous recombination comprising
the terminator region of the desired target gene from genomic
RNA Imaging with Csy4
171