2 Materials
2.1 Plasmids 1. pmax pona 12xTRICK 24xMS2SL (Addgene #64542, [25]).
- Potef-mCherry-cdc3-16boxB-3^0 UTR (Addgene #86465,
[13]). - phage ubc nls ha pcp gfp (Addgene #64539, [25]).
- Potef-λN*-3xGfp-NLS (Addgene #86780, [18]).
- pST4 TET CMV intron renilla 24xPP7 24xMS2 (Addgene
#84444, [25]). - phage UBC NLS-HA-2xMCP-tagRFP (Addgene #64541, [25]).
2.2 PCR Oligos
and Cloning
Underlined sequence represents target specific portion of the PCR
oligos, sequence highlighted in bold shows the newly introduced
restriction site.- PP7loops-SacII-Fwd:
50 CCGCCGCGGACACGGCCGTGTATTA. - PP7loops-SacII-Rev:
50 CCGCGGGCTGATCCACTCGAGAGATC. - PP7CP-BamHI-Fwd:
50 GGCGGATCCATGTCCAAAACCATCGTTCTTTCGG. - PP7CP-BamHI-Rev:
50 CATGGATCCTGAACGGCCCAGCGGCACAAGG
TTGACG.
5.BamHI and SacII restriction enzymes (e.g., New England
Biolabs).
2.3 Working
Solutions for U. maydis
[31, 32]
- Trace elements: 0.06 w/v % H 3 BO 3 , 0.14 w/v % MnCl 2 ·4
H 2 O, 0.4 w/v % ZnCl 2 , 0.4 w/v % Na 2 MoO 4 ·2 H 2 O,
0.1 w/v % FeCl 3 ·6 H 2 O, 0.04 w/v % CuSO 4 ·5 H 2 O, add
ddH 2 O, sterile filter. - Salt solution: 16 w/v % KH 2 PO 4 ,4w/v%Na 2 SO 4 , 8 w/v %
KCl, 1.32 w/v % CaCl 2 , 8 v/v % trace elements, 1 w/v %
MgSO 4 (water free), add ddH 2 O, sterile filter. - Vitaminsolution:0.1w/v%thiaminehydrochloride,0.05w/v%
riboflavin, 0.05 w/v % pyridoxine, 0.2 w/v % calcium panto-
thenate, 0.05 w/v % p-aminobenzoic acid, 0.2 w/v % nicotinic
acid, 0.2 w/v % choline chloride, 1 w/v %myo-inositol, add
ddH 2 O, sterile filter. - Complete medium (CM): 0.25 w/v % casamino acids, 0.1 w/v %
yeast-extract, 1.0 v/v % vitamin solution, 6.25 v/v % salt solu-
tion, 0.05 w/v % herring sperm DNA, 0.15 w/v % NH 4 NO 3 ,
add deionized water, adjust pH with 5 M NaOH to 7.0, add
glucose (CM-glc) or arabinose (CM-ara) solution after auto-
claving (1% f.c.).
322 Sabrina Zander et al.