The yeast one-hybrid assay was performed according to the manufacturer’s protocol in the “Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual” (Clontech). Briefly, the pTATA-box-pAbAi vector was transferred into the yeast strain and grown on medium lacking uracil (Ura). We used different concentrations of aureobasidin A (AbAr) to test the bait strain in a medium lacking Ura. The vectors of pGADT7:OsTBP2.1 were then transferred to the strains with pTATA-box-pAbAi, and the strains were grown on a medium lacking leucine with 800 ng mL−1 AbAr.
Matchmaker gold yeast one hybrid library screening system user manual
Manufactured by Takara Bio
Sourced in United States
The Matchmaker® Gold Yeast One-Hybrid Library Screening System User Manual provides instructions for the use of the Matchmaker® Gold Yeast One-Hybrid Library Screening System, a tool for identifying protein-DNA interactions.
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6 protocols using matchmaker gold yeast one hybrid library screening system user manual
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Yeast One-Hybrid Screening Protocol
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Yeast One-Hybrid Assay for ZmES22-GIF1 Interaction
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Yeast one-hybrid assays were implemented originally according to the Matchmaker® Gold Yeast One-Hybrid Library Screening System User Manual (Clontech). To test the ability of ZmES22 to bind to the core motif CATGT of GIF1 promoter, CATGT and mutant tandem repeats were cloned and inserted into the BamHI and HindIII site of the p53/AbAi vector. Yeast Y1HGlod was transformed with the vector pGADT7-ZmES22 and CATGT or mutant tandem repeats plasmids. To evaluate interaction between ZmES22 and the core motif CATGT of GIF1 promoter, the transformants were screened by plating on SD /-Leu/AbA plates.
3
Yeast One-Hybrid Assay for PpABCC1 Regulation
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The yeast one-hybrid assay (Y1H) was conducted using the Matchmaker® Gold Yeast One-Hybrid Library Screening System User Manual (Clontech, Palo Alto, CA, USA) kit. The promoter sequence of PpABCC1, 2 kb upstream of the start codon, was amplified and inserted into the reporter vector pAbAi to generate pBait-pAbAis constructs. The full-length coding sequence of the PpMYB10.1 was inserted into the effector plasmid of pGADT7 to generate the pGADT7-PpMYB10.1 construct. Positive yeast cells were used to determine the activity of the pGADT7-TF with pBait-pAbAis on SD/-Ura/AbA* medium (*, the minimal inhibitory concentration of AbA). Primer sequences used for Y1H are listed in Table S10 .
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Yeast One-Hybrid Assay for Ehd1 Promoter Analysis
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Yeast one‐hybrid assay was performed following the ‘Matchmaker Gold Yeast One‐Hybrid Library Screening System User Manual’ (Clontech, Mountain View, CA, USA). Briefly, the different parts of the Ehd1 promoter were cloned into the pAbAi vector and SIP1 was fused with pGADT7 (AD‐SIP1). Then, the proEhd1‐pAbAi vectors were transferred into the yeast strain and grown on synthetic defined medium lacking uracil (Ura). The minimal inhibitory concentration of Aureobasidin A (AbA) for the bait strain was tested on medium lacking Ura. Then, AD‐SIP was transferred into bait strains containing different proEhd1‐pAbAi vectors. The protein–DNA interactions were examined on medium lacking leucine (Leu) and Ura with 250 ng ml−1 AbA.
5
Yeast One-Hybrid Screening of GhTCE1
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Y1H screening was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual (Clontech, PT4087-1). The full-length sequence of GhTCE1 was cloned into the pGADT7 vector and transformed into yeast strain Y187, the promoter sequences of LTP2pro and LTP3pro were cloned into the pHis2 vector and transformed into yeast strain AH109, and the pGAD-empty vector was used as negative control vector. Ten microliters of each for combined yeast strains Y187 and AH109 were cultured in 200 µL YPDA liquid medium (1 L YPDA contains 10 g yeast extract, 20 g tryptone, 20 g glucose, 100 mg adenine, fill ddH2O to 1 L, pH = 6.5) at 30°C for 20–24 h. Then, 6 µL yeast that mated successfully was spotted onto SD-Trp-Leu-His containing 200 mM 3-AT solid medium at 30°C for 3–5 days, then analyzed the interaction between GhTCE1 and LTP2pro and LTP3pro. The primers used for vector construction are listed in Supplemental File S1 .
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Yeast One-Hybrid Assay for Protein-DNA Interactions
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Y1H assays were carried out by following the Matchmaker® Gold Yeast One-Hybrid Library Screening System User Manual (Clontech, USA). The recombinant plasmid p30bpTR-AbAi was transformed into yeast (Y1H Gold strain) using the Yeastmaker™ Yeast Transformation System 2 (Clontech, USA) and grown on SD/-Ura media at 30 °C for 3 days. The transformed colonies were screened by colony PCR using Matchmaker Insert Check PCR Mix 1 (Clontech, USA). Y1H assays were performed by following the Matchmaker Y1H user manual (Clontech, USA). The minimum inhibitory concentration of aeurobasidin A (AbAi) for yeasts carrying 30bpTR promoter bait was 150 ng/mL. The prey constructs (pGADT7-GOI) were transformed into yeast carrying a promoter bait fragment, plated on SD/-Leu/AbAi150, and incubated at 30 °C for 3–5 days.
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