In order to construct plasmids for yeast two-hybrid analyses, the coding sequences of AtABI5, AtRAPTOR1B, AtS6K1, AtS6K2, and AtBIN2 genes were amplified from the cDNA of Arabidopsis. The AtABI5, AtABI5-N, and AtABI5-C were inserted into the vector pGADT7. AtRAPTOR1A, AtRAPTOR1B, AtS6K1, AtS6K2, and AtBIN2 genes were cloned into vector pGBKT7. The pairs of yeast two-hybrid plasmids were co-transformed into Saccharomyces cerevisiae strain Y2H Gold following the PEG/LiAc transformation protocol (Clontech, Cat. no. 630489). In addition, the plasmids pGBKT7-53 and pGADT7 served as a positive control. The pGBKT7-Lam and pGADT7 plasmids were used as negative control. Transformants were grown at 28°C for 5 days on synthetic medium lacking Leu and Trp, then yeast colonies were transferred to synthetic medium stripped of His, Leu, Ade, and Trp containing 40 μg/mL X-a-Gal and 200 ng/mL Aureobasidin A as described by the manual (Clontech, Cat. no. 630489). Three independent experiments were performed.
Saccharomyces cerevisiae strain y2hgold
Manufactured by Takara Bio
Sourced in United States
Saccharomyces cerevisiae strain Y2HGold is a laboratory yeast strain used in yeast two-hybrid assays. It is designed to detect protein-protein interactions.
Automatically generated - may contain errors
Lab products found in correlation
Pgadt7, by Takara Bio (4 mentions)
Pgbkt7, by Takara Bio (3 mentions)
Drop out yeast media, by Takara Bio (2 mentions)
Matchmaker gold yeast two hybrid system, by Takara Bio (2 mentions)
Aureobasidin a, by Takara Bio (1 mentions)
Peg liac transformation protocol, by Takara Bio (1 mentions)
Pgbdt7, by Takara Bio (1 mentions)
Yeastmaker yeast transformation system 2, by Takara Bio (1 mentions)
Sdc leu trp, by Formedium (1 mentions)
18 protocols using saccharomyces cerevisiae strain y2hgold
1
Yeast Two-Hybrid Plasmid Construction
2
Yeast One-Hybrid Promoter Analysis
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The 2.5 kb sequences upstream of the start codon were used for the promoter analysis. Putative NRE motifs were identified using sequences revised in several recent studies [12 (link),13 (link)] and MAST algorithms available on the MEME suite server (version 5.1.0, https://meme-suite.org/meme/tools/meme (accessed on 1 September 2023)). The transformation of the Saccharomyces cerevisiae strain Y2HGold (Clontech) was performed as described in [38 (link)]. A yeast one-hybrid assay was performed as described in [39 ].
3
Transcriptional Activation of MYB Genes in Yeast
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The transcriptional activation of CsMYB45, CsMYB46, and CsMYB105 was determined in the yeast system. The ORFs of CsMYB45, CsMYB46, and CsMYB105 were constructed in the pGBKT7 vector using BamHI, resulting in the plasmids pGBKT7-CsMYB45, pGBKT7-CsMYB46, and pGBKT7-CsMYB105. pCL1 (positive control), and pGBKT7 (negative control) plasmids were introduced into Saccharomyces cerevisiae strain Y2H Gold (Clontech), in accordance with the protocol of the manufacturer (Table S1 ). The transformants harboring the pGBKT7-CsMYB45, pGBKT7-CsMYB46, and pGBKT7-CsMYB105 or pGBKT7 plasmids were selected on SD/-Trp medium (SD, Synthetic Dropout), whereas those harboring pCL1 were selected on SD/-Leu medium. As a positive control, Y2H cells harboring pCL grew well on SD/-His-Ade medium, but the negative control Y2H cells harboring pGBKT7 did not grow on SD/-His-Ade medium.
4
Yeast One-Hybrid Promoter Analysis
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The 2.5 kb sequences upstream of the start codon were used for the promoter analysis. Putative NRE motifs were identified using sequences revised in several recent studies [12 (link),13 (link)] and MAST algorithms available on the MEME suite server (version 5.1.0, https://meme-suite.org/meme/tools/meme (accessed on 1 September 2023)). The transformation of the Saccharomyces cerevisiae strain Y2HGold (Clontech) was performed as described in [38 (link)]. A yeast one-hybrid assay was performed as described in [39 ].
5
Yeast Two-Hybrid Screening for Rv3354 Interactors
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The Rv3354 gene was cloned in frame with the GAL4 DNA binding domain of pGBKT7. The resultant pGBKT7:Rv3354 vector was transformed into Saccharomyces cerevisiae strain Y2HGold following the manufacturer’s instructions (Clontech). The normalized yeast two-hybrid universal human library, fused with the GAL4 activation domain of the pGADT7 vector and stored in the Y187 yeast strain, was purchased from Clontech. The interaction between pGBKT7-53 and pGADT7-T served as a positive control, whereas pGBKT7-lam and pGADT7-T were used as a control for a negative interaction. One milliliter of the library was combined with 4 ml of a bait yeast strain and grown in 2× yeast extract-peptone-dextrose-adenine liquid medium containing 50 µg/ml kanamycin at 30°C for 24 h with slow shaking (50 rpm). Zygotes were plated on double (SD-Leu/-Trp), triple (SD-His/-Leu/-Trp), and quadruple (SD-Ade/-His/-Leu/-Trp) dropout agar plates with or without 20 mg/ml of X-α-Gal and 125 ng/ml aureobasidin. Colonies that turned blue were PCR amplified using the Matchmaker Insert Check PCR Mix 2 system (Clontech), and resulting products were sequenced at the CGRB facility of Oregon State University.
6
Constructing PhEIL2 Deletion Mutants
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The deletion mutants of PhEIL2 were constructed by PCR. The sequences of all primers used for various PhEIL2 deletion mutants are described in Supplementary Table S4 . The PCR products were fused in frame to the yeast GAL4 DNA-binding domain expression vector pGBKT7. The constructed vectors were transformed into Saccharomyces cerevisiae strain Y2HGold (Clontech, Palo Alto, CA, USA). The β-galactosidase assay was performed according to the kit instructions (Clontech).
7
Yeast Two-Hybrid Binding Assay
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The recombinant plasmids were co-transformed into Saccharomyces cerevisiae strain Y2H Gold (Clontech, Mountain View, CA, USA). Transformants were grown on synthetic medium lacking Trp and Leu (SD/-2) in an incubator at 30°C for 72 h. And then the yeast transformants were selected on medium lacking Trp, Leu, Asp, and His (SD/-4) and containing 50 mM 3AT to test binding activity.
8
Yeast Two-Hybrid Analysis of RsMYB28 and RsMYB29
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The ORF of RsMYB28 and RsMYB29 were inserted into the NcoI/SmaI and EcoRI/ BamHI cloning sites of the yeast expression vector pGBKT7 to produce pBD-RsMYB28 and pBD-RsMYB29, respectively. Both the controls (positive control: pCL1 and negative control: pGBKT7) and the recombinant plasmids were transformed into Saccharomyces cerevisiae strain Y2HGold (Clontech, Palo Alto, CA, USA) (Gao et al., 2012) (link). pCL1 transformants were selected on SD/-Leu medium, while the other transformed yeast strains were plated on SD/-Trp medium. After 3 days, all transformed cell lines were streaked onto SD/-His-Ade and SD/X-α-gal, to investigate the growth response.
9
Yeast Two-Hybrid Protein Binding Assay
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The recombinant plasmids were co-transformed into Saccharomyces cerevisiae strain Y2HGold (Clontech). Transformants were grown on the synthetic medium lacking Trp and Leu (SD/-2) at 30°C for 72 h. The yeast transformants were then selected on SD/-Trp/-Leu/-Ade/-His (SD/-4) medium for analysis of protein binding activity.
10
Yeast Two-Hybrid Assay for PMA3 Interaction
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The Gal4 DNA binding domain‐fused 14‐3‐3 or CCA309 was constructed using pGBKT7 (PT3248‐5; Clontech, Palo Alto, CA, USA) and introduced into Saccharomyces cerevisiae Y2HGold strain (Clontech) by selection with SD/‐Trp. The Gal4 activation domain‐fused fragments of PMA3 (N, M or C) were constructed using pGADT7 (PT3249‐5; Clontech) and introduced into the Y187 strain by selection with SD/‐Leu. T‐pGADT7 was used as a negative control for the interaction. Cotransformants containing pGBKT7 and pGADT7 were selected on plates lacking leucine and tryptophan (DDO). Successfully transformed yeast cells were suspended in water, and 10 µl droplets of 10‐fold serial dilutions were spotted onto DDO or SD/‐Leu/‐Trp/‐His (TDO) plates containing 100 ng ml−1 Aureobasidin A (TDO/A). The plates were incubated at 30°C for 4 d (DDO) or 6 d (TDO/A).
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