The yeast one-hybrid assay was performed as described in the Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech). Briefly, the full-length of SlbHLH59 ORF sequence (amplified from TS-186 cDNA) and promoter sequences of SlPMM, SlPMI, SlGME1, SlGMP1, SlGMP2, SlGMP3 and SlGMP4 (amplified from TS-186 genomic DNA) were cloned into the pGADT7 and pAbAi vector (Clontech), respectively. The pAbAi bait vectors were introduced into the GOLD1 yeast and cultured on SD/–Ura. The pGADT7 prey vector was introduced into yeast strains containing pAbAi bait vectors and cultured on SD/–Leu. After 4 d incubation, the positive yeast strains were picked and diluted in double-distilled water to an OD600 of 0.1, and 2 μL of suspension was spotted on SD/–Leu, with or without ABA (0–20 ng/mL) (Sigma-Aldrich), followed by 3 to 7 d incubation at 30°C.
Matchmaker one hybrid library construction and screening kit
Manufactured by Takara Bio
The Matchmaker One-Hybrid Library Construction and Screening Kit is a laboratory tool designed for the construction and screening of one-hybrid libraries. It provides the necessary components and protocols for the creation and analysis of these libraries.
Automatically generated - may contain errors
Lab products found in correlation
Pgadt7 rec vector, by Takara Bio (3 mentions)
Pabai vector, by Takara Bio (2 mentions)
Pabai br yeast integrating vector, by Takara Bio (2 mentions)
Pgadt7, by Takara Bio (1 mentions)
Matchmaker insert check pcr mix 1, by Takara Bio (1 mentions)
Matchmaker insert check pcr mix 2, by Takara Bio (1 mentions)
Pgadt7 vector, by Takara Bio (1 mentions)
Y1h gold yeast, by Takara Bio (1 mentions)
Aureobasidin a aba, by Merck Group (1 mentions)
7 protocols using matchmaker one hybrid library construction and screening kit
1
Yeast One-Hybrid Assay for SlbHLH59 Transcription Factor
2
Yeast-based Transcription Factor Assay
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The promoter of AtFT (1907 bp) was PCR amplified and inserted into a pAbAi-BR yeast integrating vector (bait-reporter; Clontech, Palo Alto, Santa Clara, CA, USA) and the AaZFP3 gene was cloned into the pGADT7-rec vector (Clontech). The pAbAi-ProAtFT and pGADT7-AaZFP3 vectors were cotransformed into the yeast Y1HGold strain using a Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech). Cotransformed yeast cells were selected on SD/-Ura-Leu (a synthetic dropout medium lacking Ura and Leu) with or without AbA (0.6 mg L−1; Sigma-Aldrich, St. Louis, MO, USA), and were then incubated for 3–5 days at 30 °C. The positive control (pGAD-p53 + p53-pAbAi) was carried out in the same manner. The primers used for these analyses are listed in Supplementary Table S2 .
3
Yeast One-Hybrid Assay for Transcriptional Activation
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Yeast one-hybrid (Y1H) assays were performed according to the manufacturer’s instruction of Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech). The safflower cDNA library cloned in the prey vector pGAD-T7 (AD) was made by the OE BioTech. In brief, the promoter fragment of CtACO3 (Figure 4A ) was cloned into the pAbAi-bait vector, which was introduced into the yeast strain Y1H GOLD, and were cultured on SD/–Ura medium. Positive clones were sequence-verified by Matchmaker Insert Check PCR Mix 1 (Clontech), the yeast-based transcriptional activation test was followed. The screen was performed by using pAbAi-bait Y1H stain and the safflower cDNA-pGADT7-DEST library. These yeast strains were cultured on SD/–Leu medium containing 100 ng/ml AbA (Clontech). Positive clones were diluted and spotted on SD/–Leu medium containing 250 ng/ml AbA (Clontech), then sequence-verified by Matchmaker Insert Check PCR Mix 2 (Clontech).
4
Yeast One-Hybrid Assay for Tomato Gene Regulation
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A yeast one-hybrid assay was carried out via a Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech, http://www.clontech.com/ ) according to the manufacturer’s instructions. A 2-kb promoter fragment of SlGME1 from the initiation site, as well as a mutated fragment of the SlGME1 promoter were amplified from tomato genomic DNA and then cloned into a pAbAi vector (Clontech). The full-length and truncated NFYA CDSs were amplified from tomato complementary DNA (cDNA) via PCR and cloned into a pGADT7 vector (Clontech); the primer sequences used are listed in Supplementary Table S2 . Both the pHIS2.0 bait vector and the pGADT7 prey vector were introduced into Y1H Gold yeast (Clontech) and cultured on SD/-Leu-Ura media. After 3 days, the positive yeast strains were selected and diluted in sterilized distilled water to an OD600 of 0.1, and 2.5 μl of suspension was spotted onto SD/-Leu-Ura media, with or without AbA (Clontech). The plates were subsequently incubated for 3–7 days at 30 °C.
5
Yeast One-Hybrid Assay for Protein-DNA Interactions
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Yeast one-hybrid assays were performed using a Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech) in accordance with the manufacturer’s instructions. In brief, a 3× promoter fragment of GhREV-08 was cloned into the pAbAibait vector, which was subsequently introduced into the yeast strain Y1H GOLD. Positive clones were cultured on SD/–Ura medium. The CDS of GhKNL1 was cloned into the pGADT7 vector, which was subsequently transformed into yeast strains containing the pAbAi bait vector; these yeast strains were cultured on SD/–Leu medium. Positive clones were diluted and spotted on SD/–Leu medium containing 600 ng ml–1 AbA (Sigma-Aldrich).
6
Yeast One-Hybrid Assay for Transcriptional Interactions
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The PsiYAB11, PtoYAB11 PSC , and PtoNGAL1 genes were cloned into the pGADT7-rec vector (Clontech, Mountain View, CA, USA) with the GAL4 activation domain. The promoters of PtoNGAL1, PtoCUC2, and PtoRBCL were inserted into a pAbAi-BR yeast-integrating vector (Clontech), which was used as a bait-reporter construct. pGADT7-PsiYAB11, pGADT7-PtoYAB11 PSC , pGADT7-PtoNGAL1, and pAbAi-Pro (PtoNGAL1, PtoCUC2, and PtoRBCL) were co-transformed into the Y1HGold yeast strain using a Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech). The full-length CDS of PsiYAB11 was inserted into the pCzn1 vector (Zoonbio, Nanjing, China) to produce recombinant PsiYAB11 in Escherichia coli BL21. The His-PsiYAB11 fusion protein was purified using PureCube Ni-NTA Agarose (Cube Biotech, Monheim, Germany). Reactions were incubated at 25°C for 30 min after adding 2 mg of protein from the His purification eluate. The primers and probes used for these analyses are listed in Supplemental Table 7 (Supplemental Methods 11).
7
Yeast One-Hybrid Assay for Plant Transcription Factor Interactions
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The PsiYAB11 and PtoYAB11 PSC genes were cloned into the pGADT7-rec vector (Clontech, Mountain View, CA, USA) with the GAL4 activation domain. The promoters of PtoNGAL1, PtoNGAL1, PtoCUC2, PtoRBCL, PtoATPA, PtoATPE and PtoPSBB were inserted into a pAbAi-BR yeast-integrating vector (Clontech), which was used as a bait-reporter construct. pGADT7-PsiYAB11, pGADT7-PtoYAB11 PSC , and pAbAi-Pro (PtoNGAL1, PtoNGAL1, PtoCUC2, PtoRBCL, PtoATPA, PtoATPE and PtoPSBB) were co-transformed into the Y1HGold yeast strain using the Matchmaker One-Hybrid Library Construction and Screening Kit (Clontech), respectively. Co-transformed yeast cells were selected on synthetic dextrose minimal medium (SD) lacking uracil (Ura) and leucine (Leu) (SD/-Ura-Leu), with or without 0.6 mg/L Aureobasidin A (AbA, Sigma-Aldrich, St. Loui, MO, USA), and were then incubated for 5 days at 30°C. pGAD-p53+p53-pAbAi and pGADT7-AD+ pAbAi-Pro were carried out in the same manner as for the positive and negative controls, respectively. The primers used for these analyses are listed in Supplemental Table S1 (Supplemental Methods S11).
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