9. DpnI restriction enzyme and 10Â buffer.
10. PCR cleanup kit.
11. NEBuilder HiFi DNA Assembly Master Mix (NEB E2621S).
12. E. coli chemically competent cells (any standard cloning strain
suitable).
13. LB liquid medium (see Subheading 2.1, item 11).
14. LB liquid medium (see Subheading 2.1, item 11) containing
100 μg/mL ampicillin.
15. LB agar plates (see Subheading 2.1, item 12) containing
100 μg/mL ampicillin.
16. 37
C Incubator.
17. 37
C Shaking incubator.
18. Plasmid miniprep kit.
2.7 Construction of
the 12xcsy Stem-Loop
Knock-In Cassette for
RNA Imaging in N.
crassa
1. pTRA.PVX.12xcsy.mCherry-2A-CP (see Subheading 3.2),
template for 12xcsy stem-loops.
2. N. crassa strain FGSC6103 genomic DNA, purified by gDNA
quick extraction protocol [22].
3. pLS3 [20], template for hygromycin B resistance cassette.
4. pAL12-Lifeact [23], template for AmpR-ORI plasmid
backbone.
5. Oligonucleotide primers for PCR-based recombinational cloning: NC-xxx-IF-F (5
0 -CCTGATTCTGTGGATAACCGTTAA
TTAA-[target gene-specific sequence]-3
0 ); NC-xxx-IF-R (5
0 -G
TGAACGCTAGCTGGTGCTGACCTCT-[target gene-specific
sequence]-3
0 ); 12xcsySL-F (5
0 -AGAGGTCAGCACCAGCTA
GC-3
0 ); 12xcsySL-R (5
0 -GGCCGGATCGATGCTAG-3
0 );
NC-Txxx-IF-F (5
0 -GCAGCTAGCATCGATCCGGCC-[target
gene-specific sequence]-3
0 ); NC-Txxx-IF-R (5
0 -GCCATATTGA
TGTAAGGTAGCTCTC-[target gene-specific sequence]-3
0 );
Pgapdh-F (5
0 -GAGAGCTACCTTACATCAA-3
0 ); Tgapdh-R
(5
0 -GGTACTATGGCTTAGATGG-3
0 ); NC-xxx-3
0 flank-F (50 -GGTATTCCATCTAAGCCATAGTACC-[target gene-specific
sequence]-3
0 ); NC-xxx-3
0 flank-R (5
0 -GTTATTGTCTCATGAG
CGGATACTTAATTAA-[target gene-specific sequence]-3
0 );
AmpR-F (5
0 -GTATCCGCTCATGAGACAATA-3
0 ); ORI-R
(5
0 -CGGTTATCCACAGAATCAG-3
0 ). PacI sites italicized (see
Subheadings 3.7 and 3.10).
6. High-fidelity (HiFi) DNA polymerase.
7. 10 mM dNTP mixture.
8. PCR thermocycler.
9. Components for agarose gel electrophoresis (see Subheading
2.2, item 11).
RNA Imaging with Csy4
163