The transcriptional activity of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 was determined by yeast one-hybrid assay using the pGBKT7 vector and the Y2HGold yeast strain (Takara Bio USA, San Jose, CA, USA). The pGBKT7 plasmid contains the GAL4 DNA-binding domain (BD) under the control of the ADH1 promoter and the TRP1 nutritional marker for selection in yeast, whereas the yeast Y2HGold strain contains the reporter genes AUR1-C, ADE2, and HIS3. The growing conditions used for the yeast Y2HGold strain are described in the Matchmaker® Gold Yeast Two-Hybrid System User Manual (Takara Bio USA). The CDSs of MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70 cDNAs were PCR amplified using primers designed for In-Fusion® cloning (Table S1 ) and cloned into the pGBKT7 vector by recombination using the In-Fusion® HD Cloning Plus kit (Takara Bio USA) following the manufacturer’s instructions. The generated constructs were sequenced as described above and then used to transform the Y2HGold yeast strain, as described in the Matchmaker® Gold Yeast Two-Hybrid System User Manual (Takara Bio USA). To test the transactivation activity, yeast strains harboring the recombinant and empty plasmids were streaked on synthetic dropout (SD) medium (-Trp, -His, -Ade) or YPDA medium supplemented with 200 ng/mL aureobasidin A. We used the papaya ERF transcription factor CpERF7 as a positive control [41 (link)].
Matchmaker gold yeast two hybrid system user manual
Manufactured by Takara Bio
Sourced in United States
The Matchmaker Gold Yeast Two-Hybrid System User Manual provides instructions for the Matchmaker Gold Yeast Two-Hybrid System, which is a tool used to study protein-protein interactions in yeast cells. The manual includes information on the system's components, experimental procedures, and troubleshooting.
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Y2hgold, by Takara Bio (2 mentions)
Matchmaker gal4 two hybrid system 3 libraries, by Takara Bio (1 mentions)
Pgadt7 vector, by Takara Bio (1 mentions)
In fusion dry down pcr cloning kit, by Takara Bio (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Yeastmakertm yeast transformation system 2 user manual, by Takara Bio (1 mentions)
Yeast protocols handbook, by Takara Bio (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Yeastmaker yeast transformation system 2, by Takara Bio (1 mentions)
Matchmaker insert check pcr mix, by Takara Bio (1 mentions)
Y2hgold yeast strain, by Takara Bio (1 mentions)
In fusion hd cloning plus kit, by Takara Bio (1 mentions)
Lysis buffer for microorganism to direct pcr, by Takara Bio (1 mentions)
Matchmaker insert check pcr mix 2, by Takara Bio (1 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Yeastmaker yeast transformation system 2 user manual, by Takara Bio (1 mentions)
Matchmaker gold yeast two hybrid system kit, by Takara Bio (1 mentions)
Oligotex mrna mini kit, by Qiagen (1 mentions)
29 protocols using matchmaker gold yeast two hybrid system user manual
1
Yeast One-Hybrid Assay of WRKY Transcription Factors
2
Identification of BpLEAP-2-interacting Proteins
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To identify BpLEAP-2-interacting MO/MФ protein(s), we screened the mudskipper MO/MФ cDNA library by yeast mating. Mating between Y2HGold/pGBKT7-BpLEAP-2 and mudskipper MO/MФ cDNA library was performed according to the Matchmaker Gold Yeast Two-Hybrid System User Manual (Clontech). After 24 h of mating, the mating yeasts were plated on SD/-Leu/-Trp supplemented with X-α-Gal and Aureobasidin A (DDO/X/A) agar plates, then cultured at 30 °C for 5 d. All blue colonies that grew on DDO/X/A were patched to high-stringency SD/-Ade/-Leu/-Trp/-His supplemented with X-α-Gal and Aureobasidin A (QDO/X/A) agar plates, then cultured for 5 d at 30 °C. Finally, 30 potential positive colonies were obtained through screening.
3
Yeast Two-Hybrid Screening of OsMADS16 Interactors
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For screening of the proteins interacting with OsMADS16, the bait strain of pGBKT7-OsMADS16 was combined with yeast library cell in 2× YPDA liquid medium using yeast mating method followed by incubation at 30°C for 21–24 h according to the Matchmaker® Gold Yeast Two-Hybrid System User Manual (Clontech, Cat. No.630489). The mating culture was plated on SD/-Leu/-Trp/-His agar plates for 7–14 d, and all the colonies were patched out onto higher stringency SD/-Leu/-Trp/-His/Ade agar plates. To further verify the interaction between them, the AD-Prey plasmids were rescued from yeast strain and sequenced. After eliminating false reading proteins, the full-length CDS of candidate proteins were amplified and cloned into prey vector pGADT7. Finally, yeast competent cell AH109 was cotransformed with the prey vector of each candidate OsMADS16-interacting protein and pGBKT7-OsMADS16, and selected on SD/-Leu/-Trp/-His and higher stringency SD/-Leu/-Trp/-His/Ade plates. The primers used are listed in S1 Table .
4
Yeast Two-Hybrid Screening Protocol
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The yeast strains Y2H-Gold and Y187 were transformed with pGADT7 and pGBKT7 vectors, respectively, by standard Lithium acetate-Tris-EDTA transformation and transformants were selected on SD-Leu or SD-Trp plates. The yeast two-hybrid was performed according to the Matchmaker® Gold Yeast Two-Hybrid System User Manual (Clontech) with a few modifications. For yeast mating, individual yeast colonies were inoculated into 5 ml of YPDA in 250 ml Erlenmeyer flasks in the morning. In the mid/late afternoon, yeast growth was measured by a spectrophotometer to ensure similar culture density. The desired mating combinations were mixed with equal amounts of media in a 500 ml Erlenmeyer flask and then the volume was brought up to ~10 ml for incubation overnight at 30°C. After 20 hours, cultures were examined for the presence of zygotes in the media and plated on SD-Trp-Leu media to select for successful mating. Following successful mating, colonies were streaked on SD-Leu-Trp-His-Ade plates supplemented with X-α-Gal and Aureobasidin A for yeast two-hybrid analysis.
5
Yeast Two-Hybrid Screening and Validation
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The coding sequence of CsTFL1 was cloned into the pGBKT7 to construct the bait vector BD-CsTFL1. A normalized cucumber Mate & Plate library was constructed by Shanghai OE Biotech using equal amounts of cDNA obtained from leaf, shoot tip and flowers of the cucumber inbred line 9930. The bait BD-CsTFL1 was used to screen the Mate & Plate library according the Matchmaker Gold Yeast Two-Hybrid System User Manual (Clontech). The full-length coding sequence of the identified interacting protein was cloned into the pGADT7. The resultant pGADT7 vector and pGBKT7 or BD-CsTFL1 were co-transformed into the Y2HGOLD yeast strain and selected on SD--Ade/-His/-Leu/-Trp to verify the interaction. In addition, full-length coding sequences for CsTFL1, CsTFL1m, CsFT, CsFD, CsFDP, Cs14-3-3-3 and Cs14-3-3-5 were cloned into pGADT7 (prey) or pGBKT7 (bait) vectors, sequenced and then transformed into the yeast strain AH109. The bait and prey vectors were transformed following the instructions of Matchmaker GAL4 Two-Hybrid System 3 & Libraries (Clontech). The protein interaction assay followed the methods of Ding et al. (2015b) (link). The primer information is listed in Table S4 .
6
Yeast Two-Hybrid Assay of OsbHLH107
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The CDS of OsbHLH107 and various truncated derivatives were amplified. The PCR products were inserted into the pGBKT7 and pGADT7 vectors, and these constructs were co-transformed into yeast strain AH109 as described in the Matchmaker™ Gold Yeast Two-Hybrid System User Manual (Clontech Laboratories). The yeast liquid culture was diluted to an absorbance of 0.5 at 600 nm (A600).
7
Yeast Two-Hybrid Screening of Peach Fruit Skin
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A Yeast Two-Hybrid Library was constructed using peach fruit skin. Cloning and testing bait for autoactivation and toxicity, and two-hybrid screening using yeast mating were performed according to the Matchmaker® Gold Yeast Two-Hybrid System User Manual (Clontech, Palo Alto, CA, USA). The domain-deleted form (301-542 a.a.) of PpGLK1, recombined into the pGBKT7 vector, was used for screening and the Y2H assay. The CDS of PpARF5 was cloned into the pGADT7 vector (Clontech). These two recombinant plasmids were co-transformed into Y2H Gold yeast cells, and then the cells cultured on selective medium lacking Trp and Leu (-T/-L) at 30°C. The putative transformants were transferred to selective medium lacking Trp, Leu, His, and adenine (-Leu/-Trp/-His/-Ade) with or without X-α-gal.
8
Yeast Two-Hybrid Assay for Protein-Protein Interactions
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The Y2H assay was performed according to the Matchmaker® Gold Yeast Two‐Hybrid System User Manual (Clontech, Takara Bio). Briefly, total RNA from CTV‐infected C. macrophylla stems was extracted to produce a cDNA library, which was further cloned into the yeast expression vector pAD (pGADT7) for screening prey proteins. The coding sequence of p33∆TMD was constructed in the pBD vector (pGBKT7) as a bait (Kang et al., 2017 ).
The pairwise Y2H was applied to validate the interaction between p33∆TMD and CmMLP2 as well as its truncations. The cDNAs encoding two polypeptides were cloned into pAD and pBD vectors, respectively. The vector construct pGBKT7‐53 was set as a positive control bait plasmid; pGBKT7‐Lam was used as a negative control bait plasmid; and pGADT7‐T was set as a positive control prey plasmid. Next, the respective combinations of plasmids were co‐transformed into the yeast cells and cultured on SD/‐Leu/‐Trp DO (DDO) medium. Upon cultivating the transformants at 30°C for 72 h, the independent colonies were further selected on SD/–Leu/–Trp/–His DO (TDO) medium, or SD/–Leu/–Trp/–Ade/–His DO (QDO) medium, or SD/‐Leu/‐Trp DO (DDO) medium plus 200 ng/mL Aureobasidin A and 40 µg/mL X‐a‐Gal (DDO/X/A).
The pairwise Y2H was applied to validate the interaction between p33∆TMD and CmMLP2 as well as its truncations. The cDNAs encoding two polypeptides were cloned into pAD and pBD vectors, respectively. The vector construct pGBKT7‐53 was set as a positive control bait plasmid; pGBKT7‐Lam was used as a negative control bait plasmid; and pGADT7‐T was set as a positive control prey plasmid. Next, the respective combinations of plasmids were co‐transformed into the yeast cells and cultured on SD/‐Leu/‐Trp DO (DDO) medium. Upon cultivating the transformants at 30°C for 72 h, the independent colonies were further selected on SD/–Leu/–Trp/–His DO (TDO) medium, or SD/–Leu/–Trp/–Ade/–His DO (QDO) medium, or SD/‐Leu/‐Trp DO (DDO) medium plus 200 ng/mL Aureobasidin A and 40 µg/mL X‐a‐Gal (DDO/X/A).
9
Yeast Two-Hybrid Screening of MeGRXC3 Interactors
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For screen the interaction proteins of MeGRXC3, a yeast two-hybrid assay has been performed in yeast strain Y2HGold based on the Matchmaker® Gold Yeast Two-Hybrid System User Manual (Clontech). DNA construct of MeGRXC3P65L:pGBKT7 was used as bait. The cDNA sequences of Arabidopsis TGA1, TGA2, TGA4, TGA5, and TGA7 were introduced into the pGADT7, in frame fused to GAL4 activate domain (AD). All constructs were pairwise co-transformed into yeast strain Y2HGold. The presence of transgenes was confirmed by growth on DDO (SD/−Leu/−Trp) plates. Interactions between two proteins were confirmed by growth on QDO/X/A (SD/−Ade/−His/−Leu/−Trp with 40 μg/mL X-alpha-Gal and 200 ng/mL Aureobasidin A).
10
Yeast Two-Hybrid Screening Validation
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To confirm the positive interactions in yeast, candidate genes were engineered into the pGADT7 vector using the In-Fusion Dry-Down PCR Cloning Kit (Clontech). The constructs were confirmed by sequencing with insert-specific primers. The bait and prey plasmids were transformed into AH109 and Y187 cells, respectively. Y2H experiments were performed following the standard procedure as described in the Matchmaker Gold Yeast Two-Hybrid System User Manual (Clontech). All the potential positive interactions generated blue or light blue colonies on SD/-Ade/-His/-Leu/-Trp /X-α-Gal agar plates.
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