The yeast one-hybrid assay was performed using MATCHMAKER Gold Yeast One-Hybrid Library Screening System (Clontech) and YEASTMAKER Yeast Transformation System 2 (Clontech). The amplified promoter regions were cloned upstream of the Aureobasidin A resistance (AUR1-C) reporter gene in the pAbAi vector to generate the following constructs: pAbAi-proVdSTS2ful, pAbAi-proVdSTS2del1, pAbAi-proVdSTS2del2. The VdMYB1 ORF was cloned in frame after the GAL4 transcriptional activation domain (AD) in pGADT7, and the resulting AD-VdMYB1 was co-introduced with other pAbAi vectors into the yeast strain Y1HGold. The transformed yeast cells were cultured on SD medium containing 0 or 200 ng/ml Aureobasidin A at 30 °C for 3 days. The β-galactosidase activity of positive clones was identified according to the manufacturer’s instructions.
Yeastmaker yeast transformation system 2
Manufactured by Takara Bio
Sourced in United States, Japan, China
The Yeastmaker Yeast Transformation System 2 is a laboratory equipment designed for the transformation of yeast cells. It provides a standardized and efficient method for introducing foreign DNA into yeast, a crucial step in genetic engineering and research applications involving yeast.
Automatically generated - may contain errors
Lab products found in correlation
Matchmaker gold yeast two hybrid system, by Takara Bio (11 mentions)
Pgbkt7, by Takara Bio (11 mentions)
Pgadt7, by Takara Bio (7 mentions)
Normalized universal human cdna library, by Takara Bio (4 mentions)
Y2hgold yeast strain, by Takara Bio (4 mentions)
Matchmaker gold yeast one hybrid library screening system, by Takara Bio (4 mentions)
Htx kit, by Dualsystems Biotech (3 mentions)
Make your own mate plate library system, by Takara Bio (3 mentions)
Easyclone cdna library construction kit, by Dualsystems Biotech (2 mentions)
Matchmaker insert check pcr mix, by Takara Bio (2 mentions)
Matchmaker insert check pcr mix 1, by Takara Bio (2 mentions)
X α gal, by Coolaber (2 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (2 mentions)
Matchmaker tm insert check pcr mix, by Takara Bio (2 mentions)
Sd leu aba aureobasidin a, by Takara Bio (2 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Fv1200 laser scanning microscope, by Olympus (1 mentions)
Plasmid midi kit, by Qiagen (1 mentions)
In fusion cloning kit, by Takara Bio (1 mentions)
Saccharomyces cerevisiae strain y2hgold, by Takara Bio (1 mentions)
111 protocols using yeastmaker yeast transformation system 2
1
Yeast One-Hybrid Assay for VdMYB1 Transcriptional Regulation
2
Yeast-One-Hybrid Screening of EfPAL2 Promoter
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Seed kernels (10, 20, 30 and 40 DAF) (Wu et al., 2021 (link))and leaves (diameter range 15-200 cm) (Wu et al., 2022b (link)) of E. ferox were subjected to cDNA library construction (oebiotech, Shanghai, China). The promoter of EfPAL2 (1000 bp immediately upstream of the EfPAL2 open reading frame) was reconstituted into the pAbAi vector using the restriction enzymes Kpn I and Sal I. The recombinant plasmids were linearized using restriction endonuclease BstbI. The linearized pPAL2-1000-PAbAi was transferred into YIH yeast using the Yeastmaker™ Yeast Transformation System 2 (Clontech) and grown on plates of SD/-Ura for three days. The colonies were resuspended with 0.9% NaCl, its OD600 value was adjusted to 0.002, and then coated on plates containing different concentrations (100-1000 ng/ml) of Aureobasidin A (AbA) and screened to the lowest AbA concentration that inhibited its auto-activation. The competent cell was produced with Y1HGold containing pPAL2-1000-PAbAi, then transferred to the library plasmid and incubated on SD/-Leu+AbA100 plates at 30°C for 3-5 days. Positive clones verified by PCR were sequenced and then annotated by blastx in NCBI (https://www.ncbi.nlm.nih.gov/ ). The primers used above are listed in Table S3
3
Yeast Two-Hybrid Assay for SlGCN5, SlADA2a, and SlADA2b
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To obtain yeast two-hybrid vectors, the full-length SlGCN5 was cloned into pGADT7 (Clontech). The full-length SlADA2a and SlADA2b were individually cloned into pGBKT7 (Clontech). The yeast two-hybrid assay was performed using the Yeastmaker Yeast Transformation System 2 (Clontech, T2001) according to the instruction of the manufacturer. Primer sequences are provided in Supplementary Table 1 .
4
Screening Protein-Protein Interactions Using Y2H
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Y2H screening was performed using the Matchmaker Gold Yeast Two-Hybrid System according to the manufacturer’s instructions (Clontech). To generate the bait and prey constructs, the coding sequences of SDI1, MYB28, MYB29, and MYB76 were PCR-amplified using the primers listed in table S12 and directionally cloned into the pGBKT7 and pGADT7 (Clontech) vectors using the In-Fusion HD Cloning Kit according to the manufacturer’s instructions (Clontech). One hundred nanograms of pGBKT7-SDI1 (SDI1-BD) and pGADT7-MYBs (MYB28/MYB29/MYB76-AD) vectors was introduced into Y2HGold yeast strains using Yeastmaker Yeast Transformation System 2 (Clontech). Negative (pGBKT7-Lam and pGADT7-T) and positive (pGADT7-53 and pGBKT7-Lam) controls were used according to the manufacturer’s instructions. The Y2HGold yeast strains cotransformed with pGBKT7 and MYB28/MYB29/MYB76-AD were used as additional negative controls. Yeast harboring bait and prey vectors were plated on double dropout medium containing X-α-Gal (40 μg/ml; -Leu/Trp/X = DDO/X) and quadruple dropout medium containing X-α-Gal (40 μg/ml) and Aureobasidin A (200 ng/ml; -Leu/Trp/Ade/His/X/A = QDO/X/A). To verify the growth of positive colonies, 3 days after transformation, target colonies were selected and resuspended in double-distilled water and dropped on DDO, QDO, DDO/X, and QDO/X/A.
5
Yeast Two-Hybrid System Protocol
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The YTH assay was conducted using the Matchmaker Gold Yeast Two‐Hybrid System and the Yeastmaker Yeast Transformation System 2 (Clontech, Mountain View, CA, USA) as previously described (Yang et al., 2017 ).
6
Yeast Two-Hybrid Screening of TMEM39A
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The cDNA coding sequences of the first and second cytoplasmic loop domain of human TMEM39A were cloned into the pGBKT7 vector and screened against a normalized universal human cDNA library (Clontech, 630481), following instruction of the Matchmaker Gold Yeast Two-Hybrid System (Clontech, 630489). Verification of positive colonies was achieved by co-transforming wild-type or YR-mutant TMEM39A loop domain (in pGBKT7 Vector) with genes of interest (in pGADT7 Vector) following the instruction of YeastMaker Yeast Transformation System 2 (Clontech, 630439) as well as plasmids from re-cloned cDNA.
7
Constructing Ginger Y2H Library
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For ginger library construction Y187 yeast competent cells were prepared according to the protocol of the Yeastmaker Yeast Transformation System 2 (Clontech, USA). About 20 μL of the ds cDNA of ginger (2–5 μg) and 3 μg of pGADT7 were co-transformed into the yeast strain Y187 to construct the ginger Y2H library according to the protocol mentioned in Make Your Own “Mate & Plate™” Library System (Clontech, USA). To determine the complexity of the library, 100 μL of 1/10 and 1/100 dilutions of transformed cells were spread on SD/−Leu (synthetically defined medium lacking leucine) 100 mm agar plates. After incubation at 30°C for 3–4 days, the number of colonies on dilution plates was counted and the transformation efficiency was calculated. To identify the titer of the constructed ginger Y2H library, 10 μL of library aliquot was taken out and diluted to 1/100, 1/1000, 1/10000, and 1/1000000. The last two dilutions were spread in duplicate on SD/−Leu 100 mm agar plates, and the library titer was calculated according to the colonies appearing (Fu et al., 2013 (link)).
8
GFP Tagging of NbSWP12 Protein
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For GFP tagging of NbSWP12, DNA fragment of Nbswp12 was amplified by PCR from p-Cold I-Nbswp12 plasmid with primers containing BamH I or Sal I restriction site (Table 1 ) and inserted into pUG35 plasmid. The pUG35 and pUG35-Nbswp12 plasmids were transformed into yeast S. cerevisiae CEN.PK2 using Yeastmaker™ Yeast Transformation System 2 (Clontech, Mountain View, CA, USA). Transformants were selected in Minimal synthetic defined (SD) bases with Uracil Dropout Supplement (SD/-Ura). Isolated clone was grown in SD/-Ura liquid overnight and inoculated into SD/-Ura-Met inducing medium for overexpression under control of MET25 inducible promoter. Yeasts were collected at 24 or 48 h post inoculating, and location signals were observed under Olympus FV1200 Laser Scanning Microscope.
9
Serpin-cSP Protein Interactions Validation
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The experiments were performed as previously described [52 (link)], with primers listed in S6 Table . Serpins were cloned into pGBK7 and cSPs were introduced into pGADT7 plasmid. For the validation of serpin-5 and serpin-9 binding proteins, the two types of plasmids were co-transformed into AH109 cells using YeastMaker Yeast Transformation System 2 (Clontech, Mountain View, CA, USA), and the binding was validated in synthetic dropout-Leu-Trp-His medium supplemented with X-gal.
10
Yeast Two-Hybrid Assay for OsGA2ox8 Interactors
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The coding sequence (CDS) of OsGA2ox8 was fused to the GAL4 DNA-binding domain (DNA-BD) to generate the pGBKT7-OsGA2ox8 construct. The cDNA library generated from leaves of DK151 [25 (link)] at the tillering stage was used for fusion to the GAL4 activation domain by Takara Biotechnology Co., Ltd. (Dalian, China). A yeast two-hybrid assay was performed using the Yeastmaker™ Yeast Transformation System 2 (Clontech, Mountain View, CA, USA) in accordance with the manufacturer’s instructions. Full-length fragments of the candidate interacting proteins were cloned into pGADT7 as prey to confirm the interaction. The pGBKT7-OsGA2ox8 and prey plasmids were co-transformed into yeast strain AH109 and then cultured on SD/–Ade/–His/–Leu/–Trp/X-α-gal plates. A filter assay was performed to test for β-galactosidase activity. The specific primers used to amplify each full-length fragment are listed in Supplementary Table S3 .
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