Minimal medium,10 (link)) yeast peptone dextrose broth (Takara), and Luria–Bertani (LB) broth were used to culture A. oryzae RIB40 (NBRC 100959), Saccharomyces cerevisiae Y2HGold (Takara, Shiga, Japan), and E. coli DH5α (Takara), respectively.
Saccharomyces cerevisiae y2hgold
Manufactured by Takara Bio
Sourced in United States
Saccharomyces cerevisiae Y2HGold is a yeast strain used for yeast two-hybrid (Y2H) screening. It is designed to detect protein-protein interactions and screen for novel interacting partners.
Automatically generated - may contain errors
Lab products found in correlation
Pgadt7 vector, by Takara Bio (2 mentions)
Pgadt7, by Takara Bio (2 mentions)
E coli dh5α, by Takara Bio (1 mentions)
Frozen ez yeast transformation 2 kit, by Zymo Research (1 mentions)
Yeast strain y187, by Takara Bio (1 mentions)
Matchmaker gold yeast two hybrid system, by Takara Bio (1 mentions)
Ecori, by Takara Bio (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Amv reverse transcriptase, by Takara Bio (1 mentions)
Bamhi, by Takara Bio (1 mentions)
Rnaiso reagent, by Takara Bio (1 mentions)
Takara mutanbest kit, by Takara Bio (1 mentions)
Yeast protocols handbook, by Takara Bio (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (1 mentions)
E coli dh5α, by Thermo Fisher Scientific (1 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Pgadt7 t, by Takara Bio (1 mentions)
Pgbkt7 lam, by Takara Bio (1 mentions)
Pgbkt7 53, by Takara Bio (1 mentions)
10 protocols using saccharomyces cerevisiae y2hgold
1
Cultivation of model organisms
2
Yeast Two-Hybrid Screening for Protein Interactions
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The yeast two-hybrid assays were performed in strain Saccharomyces cerevisiae Y2HGold (Takara Bio USA). Combination of the two plasmids encoding the desired fusion proteins were transformed into Y2HGold cells using Frozen-EZ Yeast Transformation II Kit (Zymo Research). Selection for growth was carried out on selective drop-out plates lacking leucine and tryptophan (SD-Leu-Trp) and single colonies were inoculated into liquid SD-Leu -Trp medium and grown overnight at 30°C. Saturated cultures were diluted 1:4 in water and 5 µL of each dilution was then spotted on SD-Leu-Trp and SD-Leu -Trp -Ade -His (additionally lacking adenine and histidine) in order to test for growth and interaction, respectively. Plates were incubated for 4–5 days at 30°C and scanned. Each interaction was tested in biological triplicate experiments.
3
Yeast Two-Hybrid Screening of HeLa cDNA Library
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The Matchmaker® Gold Yeast Two-Hybrid System (Clontech) was used to screen of a pre-transformed Mate and Plate™ Library—normalized Universal Human HeLa cDNA library (Clontech), following the instructions of the manufacturer. In summary, Saccharomyces cerevisiae Y2HGold (Clontech) was transformed with plasmid pFA147, a derivative of pGBKT7 (Table S1 ) and mated with yeast strain Y187 (Clontech) carrying the HeLa cDNA library cloned into pGADT7. The two strains were mated and plated in lower stringency or double dropout (DDO) media (SD/–Leu/–Trp) supplemented with X-α-Galactosidase (X) and Aureobasidin A (A) (all from Clontech) and incubated 3–5 days at 28–30°C. The blue colonies grown on DDO/X/A media were patched into a higher stringency or quadruple dropout (QDO) media (SD/–Ade/–His/–Leu/–Trp) supplemented with X-α-Galactosidase and Aureobasidin A and incubated 3–5 days at 28–30°C. The blue colonies that grew in QDO/X/A were further analyzed for autonomous system activation, and for identification of the target protein, by sequencing the plasmid DNA (primers listed in Table S2 ). We analyzed 1.43 × 108 clones, patched 60 blue colonies and recovered 41 from high stringency media for further analysis.
4
Cloning and Validating BpLEAP-2 Bait Plasmid
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Total RNA was extracted from the livers of healthy mudskippers using RNAiso reagent (TaKaRa, China). After treatment with DNase I (TaKaRa), first cDNA strand synthesis was carried out using AMV reverse transcriptase (TaKaRa). Two primers, i.e., LEAP-2-m1(+): 5'-CCG
GAATTC ATGACCCCTCTCTGGAGGAT-3' (EcoR I site is underlined) and LEAP-2-m1(−): 5'-CGC
GGATCC TCAGTTGGTGACAGAAACGG-3' (BamH I site is underlined), were used to amplify the sequence encoding the mature peptide (aa 55–100) of BpLEAP-2. The amplicons were subsequently digested with EcoR I and BamH I (Takara) and ligated into the EcoR I-BamH I-cleaved pGBKT7 vector (Clontech, USA) to construct the bait plasmid pGBKT7-BpLEAP-2. The pGBKT7-BpLEAP-2 plasmid and empty pGBKT7 vector were transformed into Saccharomyces cerevisiae Y2HGold (Clontech) to test the autoactivation and toxicity of the bait protein. Transformants were then grown on SD/-Trp, SD/-Trp/X-α-Gal (40 μg/mL X-α-Gal), and SD/-Trp/X-α-Gal/AbA (40 μg/mL X-α-Gal and 125 ng/mL Aureobasidin A) agar plates for 3–5 d. Only bait that lacked autoactivation and toxicity was used in Y2H screening.
5
Yeast Two-Hybrid Screen for RID1 Interactors
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The full-length RID1 cDNA and fragments of RID1 were cloned into the pGADT7 vector (Clontech, Palo Alto, CA, USA) at the EcoRI and XhoI sites to create the pAD-RID1 bait vector. RID1 fragments with mutant amino acid residues were created using a TaKaRa MutanBEST Kit (TaKaRa, Dalian, China). The pBD-GFA1 prey vector was a generous gift from Dr Yang Wei-Cai (Chinese Academy of Sciences, Beijing, China). Yeast transformation was carried out using a kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s protocol. The yeast strain used was Saccharomyces cerevisiae Y2H gold (Clontech, Palo Alto, CA, USA). Transformed cells were plated on SD-Trp-Leu/X-α-Gal/AbA for screening of positive colonies, and incubated at 30 °C for 3–5 d. Host yeast cells were transformed with the Empty AD/BD vectors used as negative controls.
6
Yeast Two-Hybrid Protein Interaction Assay
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The full-length coding sequences of genes were cloned into the pGBKT7 (BD-) vector as bait and prey pGADT7 (AD-) vector as prey. The recombinant plasmids were transformed into the yeast strain Saccharomyces cerevisiae Y2HGold (Clontech). Yeast cotransformed with the empty pGADT7 vector was used as negative control. Transformed yeast colonies were selected on synthetic dropout medium minus Leu and Trp. Protein interactions were examined on selective medium (SD-Trp-Leu-His with 5 mM 3-amino-1,2,4-triazole) for 3 d at 30 °C. The primers used for this assay were designed as described in Supplemental Table S6 .
7
Yeast Two-Hybrid Screening of BLH Transcription Factors
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The full-length coding regions of BLH6b and BLH2 were amplified from P. alba × P. glandulosa xylem cDNA with the Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) and cloned into the pGADT7 vector by using the ClonExpressII One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China), generating AD fusion vectors. The full-length coding region of BLH6a was cloned into the pGBKT7 vector, generating the BD-BLH6a vector. The BD-BLH6a and AD fusion vectors were co-transformed into the yeast strain Saccharomyces cerevisiae Y2HGold as described in the Yeast Protocols Handbook (Clontech Laboratories, Inc., Mountain View, CA, USA). Transformants were grown on the SD/-Leu-Trp medium and then screened on the SD/-Ade-His-Leu-Trp medium.
8
Microbial Strain Cultivation Protocols
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The strains were E. coli DH5α (Invitrogen) and Rosetta (Merck), P. syringae pv. tomato DC3000 (Pst) (Guo et al., 2009 ), A. tumefaciens GV2260 (Deblaere et al., 1985 ), Saccharomyces cerevisiae Y2HGold (Clontech), and B. cinerea B05.10 (Ma et al., 2017 ). Bacterial, yeast, and fungal strains were grown with the appropriate antibiotics as follows: E. coli in Luria‐Bertani (LB) medium at 37 °C, Pst and A. tumefaciens in LB medium at 28 °C, S. cerevisiae at 30 °C in selective synthetic complete medium supplemented with 2% glucose, and B. cinerea in potato dextrose broth (PDB) at 20 °C.
9
Yeast Two-Hybrid Protein Interaction Assay
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To test in vitro interaction, the coding sequence of ELF3-1 and ELF3-2 were cloned into pGADT7 vector (Clontech) whereas the coding sequence of LUX was cloned in pGBKT7 (Clontech). LUX in pGBKT7 and either ELF3 in pGADT7 vectors were used to transform Saccharomyces cerevisiae Y2HGold (Clontech). Positively transformed yeast grew in a synthetic defined medium lacking the amino acids leucine and tryptophan (SD). Interaction screening was performed in SD without adenine (-Ade) and histidine (-His).
10
Yeast Two-Hybrid Interaction Assay
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Saccharomyces cerevisiae Y2HGold (Clontech) was used for the Y2H assay. pGBKT7‐ and pGADT7‐derived constructs were transformed into Y2HGold and the Y2H experiment was performed according to the protocols provided by the manufacturer. DDO (minimal synthetic defined base with added double dropout supplement − Leu/−Trp) culture plate was used for positive transformation screening and QDO (SD base with added quadruple dropout supplement − Ade/−His/−Leu/−Trp)/X‐α‐Gal culture plate was used for protein interaction verification. Vectors pGBKT7‐53 and pGADT7‐T (Clontech) were used as positive control because the murine p53 protein (53) can interact with SV40 large T‐antigen (T) in yeast, while pGBKT7‐Lam (Clontech) and pGADT7‐T were used as negative control because human lamin C protein (Lam) cannot interact with T in yeast.
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Saccharomyces cerevisiae Y2HGold (Clontech) was used for the Y2H assay. pGBKT7‐ and pGADT7‐derived constructs were transformed into Y2HGold and the Y2H experiment was performed according to the protocols provided by the manufacturer. DDO (minimal synthetic defined base with added double dropout supplement − Leu/−Trp) culture plate was used for positive transformation screening and QDO (SD base with added quadruple dropout supplement − Ade/−His/−Leu/−Trp)/X‐α‐Gal culture plate was used for protein interaction verification. Vectors pGBKT7‐53 and pGADT7‐T (Clontech) were used as positive control because the murine p53 protein (53) can interact with SV40 large T‐antigen (T) in yeast, while pGBKT7‐Lam (Clontech) and pGADT7‐T were used as negative control because human lamin C protein (Lam) cannot interact with T in yeast.
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