S. pombe were grown in the Yeast Extract Plus Supplements (YES) and selective Edinburgh minimal medium (EMM) and Pombe glutamate medium. The YES medium contained dropout mix (2 g/liter), glucose (30 g/liter), and Difco yeast extract (5 g/liter) as previously described (25 (link)). The EMM was prepared as previously described (68 (link)). PMG was identical to EMM, except that the nitrogen source was monosodium glutamate (3.75 g/liter). When indicated, B1 (Sigma-Aldrich) was added to a final concentration of 10 μM to repress the nmt1 promoter. 5-FOA (5-fluoroorotic acid, U.S. Biologicals) was added to a final concentration of 1 mg/ml. To make YES+5-FOA + Nat plates, a final concentration of nourseothricin (100 μg/ml; ClonNAT, Jena Bioscience, Germany) and 5-FOA (1 mg/ml) was added to YES medium containing 2% agar. G418 (Geneticin, Sigma-Aldrich) was added to a final concentration of 500 μg/ml in the YES+5-FOA + G418 plates. To generate DNA damage stress, hydroxy urea (HU, Sigma-Aldrich) was added to a final concentration of 5 mM in the YES plates. All S. pombe strains were grown at 32°C.
Clonnat
Manufactured by Jena Biosciences
Sourced in Germany
ClonNat is a selective antibiotic used for the screening and maintenance of transformed cells in cell culture applications. It acts as a resistance marker, allowing for the identification and propagation of successfully transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Zeocin, by Nacalai Tesque (2 mentions)
Hygromycin, by Nacalai Tesque (2 mentions)
Hydroxyurea hu, by Merck Group (1 mentions)
Geneticin g418, by Merck Group (1 mentions)
Kanamycin, by Bio Basic (1 mentions)
Codon optimization tool, by Integrated DNA Technologies (1 mentions)
Rotor hda pinning robot, by Singer Instruments (1 mentions)
Peptone, by BD (1 mentions)
Escherichia coli novablue, by Merck Group (1 mentions)
Yeast extract, by BD (1 mentions)
Yeast nitrogen base without amino acids, by BD (1 mentions)
Yeast extract, by Nacalai Tesque (1 mentions)
Tryptone, by Nacalai Tesque (1 mentions)
Escherichia coli strain dh5α, by Toyobo (1 mentions)
Bacto peptone, by BD (1 mentions)
8 protocols using clonnat
1
S. pombe Growth and Stress Conditions
2
Optimized Antibiotic Resistance Markers for Candida
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Gene sequences were generated with Integrated DNA Technologies’ (IDT) Codon Optimization Tool, with their open reading frames first optimized to match codon usage of Candida albicans, and then edited further, as described in the Results. Genes were synthesized by GENEWIZ (South Plainfield, NJ, USA). The functionality of de novo synthesized antibiotic resistance markers was verified in E. coli at 37 °C on solid LB-Luria plates (10 g L−1 of bacto tryptone, 5 g L−1 of yeast extract, 0.5 g L−1 of NaCl, 15 g L−1 of agar) supplemented with 100 µg mL−1 of ampicillin and one of the following antibiotics: 50 µg mL−1 of kanamycin (BioBasic, Toronto, ON, Canada), 150 µg mL−1 of hygromycin B (Fisher Scientific International LLC, Waltham, MA, USA), 50 µg mL−1 of clonNAT (Jena Bioscience, Jena, Thuringia, Germany), or 20 µg mL−1 of phleomycin (Fisher Scientific International LLC, Waltham, MA, USA).
3
Synthetic Genetic Array Protocol
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Three independent biological replicates of each SGA were performed with the Bioneer V5 deletion collection40 (link) using a ROTOR HDA pinning robot (Singer Instruments) as previously described41 (link). Briefly, the three query strains h-btn1::NatMX ura-D18 leu1-32 ade6-M210, h-btn1102–208del::NatMX ura-D18 leu1-32 ade6-M210, h-btn1D363G::NatMX ura-D18 leu1-32 ade6-M210 and a control query strain, h-ade6::NatMX ura-D18 leu1-32 ade6-M21042 (link) were mated with the library on Edinburgh Minimal Medium without nitrogen (Formedium). Following sporulation for 3 days at 25 °C and spore selection for 3 days at 42 °C, spores were pinned onto YES agar for 2 days at 32 °C to recover. Double mutant haploids were selected by growing cells on YES agar with ClonNat (Jena Bioscience; AB-102XL; 100 μg/ml) and G418 (Formedium; 500 μg/ml) for 2 days at 32 °C. Double mutant libraries were then grown on YES agar in quadruplicate (1536-well format) for 2 days at 32 °C.
4
Transforming Diatom T. pseudonana
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The resulting gene constructs were introduced into T. pseudonana cells using the Biorad PDS-1000/He particle delivery system as described previously30 . Co-transformations were performed with the pTpfcp/nat plasmid DNA30 for selection of transformed cell lines on agar plates containing 150 μg/mL ClonNat (Jena Biosciences).
5
Plasmid Construction and Yeast Cultivation
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Escherichia coli NovaBlue (Merck Millipore, Darmstadt, Germany) was used for plasmid construction and amplification. The microorganisms were routinely cultured at 37 °C and 200 rpm in LB medium [10 g/L tryptone (Nacalai Tesque, Kyoto, Japan), 5
g/L yeast extract (Nacalai Tesque), and 5 g/L NaCl] supplemented with 100 μg/mL ampicillin.
The yeast strains used in this study are listed in Table 1. P. pastoris CBS7435 Δdnl4 Δhis4 was used as a parent strain since it has improved gene targeting efficiency for homologous recombination 37 (link) . P. pastoris strains were cultivated in YPG medium [10 g/L yeast extract, 20 g/L peptone (BD Biosciences, San Jose, CA, USA), and 20 g/L glycerol], YPD medium [10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose], or SD medium [6.7 g/L yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), and 20 g/L glucose] supplemented with 20 mg/L histidine and appropriate antibiotics including 500 μg/mL G418 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 300 μg/mL hygromycin (Nacalai Tesque), 100 μg/mL Zeocin (Nacalai Tesque), and 50 μg/mL clonNAT (Jena Bioscience, Löbstedter, Germany).
g/L yeast extract (Nacalai Tesque), and 5 g/L NaCl] supplemented with 100 μg/mL ampicillin.
The yeast strains used in this study are listed in Table 1. P. pastoris CBS7435 Δdnl4 Δhis4 was used as a parent strain since it has improved gene targeting efficiency for homologous recombination 37 (link) . P. pastoris strains were cultivated in YPG medium [10 g/L yeast extract, 20 g/L peptone (BD Biosciences, San Jose, CA, USA), and 20 g/L glycerol], YPD medium [10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose], or SD medium [6.7 g/L yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), and 20 g/L glucose] supplemented with 20 mg/L histidine and appropriate antibiotics including 500 μg/mL G418 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 300 μg/mL hygromycin (Nacalai Tesque), 100 μg/mL Zeocin (Nacalai Tesque), and 50 μg/mL clonNAT (Jena Bioscience, Löbstedter, Germany).
6
Yeast media and genetic selection
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Standard yeast media were used, 100 µg/ml ampicillin was added to liquid media to avoid bacterial contaminations. YPD liquid media and plates containing 300 μg/ml G418 Sulfate (Geniticin, American Bioanalytical) were used to select for KanMX or/and 100 μg/ml ClonNat (Jena Bioscience, Jena, Germay) to select for NatMX. SC media lacking leucine was used for growth of yeasts containing the complementation plasmids (V0-V4). SC media containing uracil was used for selection for growth in presence of 1 mg/ml 5-Fluoro Orotic Acid (5-FOA), to select against cells with functional URA3. SC-Glycerol and SC-Ethanol plates are used to test for mitochondrial function.
7
Yeast Strain Cultivation and Plasmid Amplification
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Characteristics of all yeast strains used in this work are summarize in Table 1. P. pastoris CBS7435 was used as a parent strain. P. pastoris strains were cultivated in YPD medium [10 g/L yeast extract, 20 g/L Bacto-peptone (Difco Laboratories, Detroit, MI, USA), and 20 g/L glucose] supplemented with 500 μg/mL G418 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 300 μg/mL hygromycin (Nacalai Tesque), 100 μg/mL Zeocin (Nacalai Tesque), and/or 50 μg/mL clonNAT (Jena Bioscience, Löbstedter, Germany) as required. Escherichia coli strain DH5α (Toyobo, Osaka, Japan) was used for construction and amplification of plasmid DNA. The medium for E. coli growth was prepared as previously described (Inokuma et al. 2016a (link)).
8
Genetic Manipulation of Fission Yeast
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For construction of strain PD13, the strains FY15592 and SI034 (Table S1) were crossed and selected on EMM with G418 (49418.04 Serva) and ClonNat (LEXSY NTC, AB-102, Jena Bioscience). To obtain Movie S1 and Figure 1A, Rec8-GFP-(FY13699, Table S1) labeled cells were crossed with cells labeled for Sid4-mCherry (SI661, Table S1), and zygotes were imaged as described below. To observe heterologous pairing, strains PD05 and PD14 were crossed and meiosis followed under the microscope. Observation of cen2-homologous loci was obtained by crossing AK03 and AK04.
Overexpression of either tubulin-mCherry or Mus81 was obtained by transforming PD13 cells via standard transformation procedure with lithium acetate, using 5 mg plasmid DNA. Plasmid pSV01 was generated in the I.M.T. lab and pREP1-RusA was kindly provided by Paul Russel. We selected transformed cells on leu-deficient medium. Cells without the plasmid did not grow on leudeficient medium (Figure S4C).
Overexpression of either tubulin-mCherry or Mus81 was obtained by transforming PD13 cells via standard transformation procedure with lithium acetate, using 5 mg plasmid DNA. Plasmid pSV01 was generated in the I.M.T. lab and pREP1-RusA was kindly provided by Paul Russel. We selected transformed cells on leu-deficient medium. Cells without the plasmid did not grow on leudeficient medium (Figure S4C).
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