AgMDL1 was amplified from Sua5B cDNA and cloned into the pGBKT7 bait vector using the primers described in S1 Table according to the Matchmaker Gold Yeast Two Hybrid System user protocol (Clontech). For cDNA library construction, total RNA was isolated from Sua5B cells, reverse-transcribed into cDNA, and cloned into the pGADT7 prey vector using the Make Your Own Mate and Plate Library System (Clontech). pGBKT7 (bait) and pGADT7 (prey) vectors were transformed in the Y2HGold and Y187 yeast strains, respectively. Screening was performed according to the Matchmaker Gold Yeast Two-Hybrid System user protocol (Clontech) using either double dropout synthetic medium (leucine and tryptophan) or quadruple dropout synthetic medium (leucine, tryptophan, adenine, histidine) supplemented with aureobasidin and X-α-Gal (Clontech). Positive clones were sequenced and blasted against the An. gambiae genome in Vectorbase. Interacting proteins were confirmed using standard pulldown assays.
Make your own mate and plate library system
Manufactured by Takara Bio
Sourced in United States
The Make Your Own Mate and Plate Library System is a laboratory equipment product designed for creating custom DNA libraries. It enables users to assemble plasmids and construct libraries of their choice. The system provides the necessary tools and components to facilitate these DNA manipulation tasks.
Automatically generated - may contain errors
4 protocols using make your own mate and plate library system
1
Yeast Two-Hybrid Screening of AgMDL1
2
Constructing Drought-Tolerant Chickpea cDNA Library
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The cDNA library was constructed from the roots of the drought tolerant chickpea genotype, ICC 4958 in S. cereviceae Y187α using Make Your Own “Mate and Plate™” Library System (Clontech, USA) following the manufacturers' instructions. Equal amounts of double stranded cDNA (3 μg) and “prey” library vector (3 μg; pGADT7-Rec) were mixed for the homologous recombination-mediated cloning using the library-scale transformation protocol (Yeast Transformation System 2 Manual, Clontech, USA). After 4 days of incubation, all the colonies were harvested in freezing medium (YPDA in 25% glycerol) and stored in aliquots at –80°C.
3
Investigating Protein-Protein Interactions of OsSalT
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Yeast two hybrid assay was performed using Matchmaker Gold Yeast Two-Hybrid System (Clontech). The OsSalT gene was cloned in-frame into the EcoRI and BamHI RE sites of the yeast vector, pGBKT7 to obtain a BD-OsSalT construct as bait. To study the protein-protein interaction of OsSalT, a cDNA library, ligated to pGADT7-Rec as prey, was prepared from rice roots under drought stress using Make Your Own "Mate and Plate" Library System (Clontech). The constructs were co-transformed into Y2H Gold yeast strain and screened on DDO (SD/-Leu/-Trp) and QDO (SD/-Ade/-His/-Leu/-Trp) plates. This was followed by a QDO/X/A (SD/-Ade/-His/-Leu/-Trp/X-α-Galactosidase/Aureobasidin-A) selection to test interaction. To identify the interacting protein partner(s), selected colonies were analysed by PCR followed by sequencing according to the manufacturer's instruction. The AD-T-antigen and BD-p53 interaction was used as a positive control.
4
Yeast Two-Hybrid Analysis of Drought Response Proteins
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Yeast two-hybrid analysis was performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech) following manufacturer's protocol. The Osr40c1 gene was cloned into EcoRI and BamHI RE sites of pGBKT7 vector to generate recombinant BD-Osr40c1 bait construct. Yeast transformation was performed according to the instructions of clonetech manual. The cDNA library was prepared from rice roots collected from the plants exposed under drought stress. The cDNA library was ligated to pGADT7-Rec vector to prepare recombinant AD construct using Make Your Own "Mate and Plate" Library system (Clontech). The yeast strain Y2H gold was co-transformed with bait and prey recombinant construct and colonies were screened against DDO (SD/-Leu/-Trp) and QDO/X/A (SD/-Ade/-His/-Leu/-Trp with aureobsidin A and X-α-gal). Selected blue colonies were analyzed by sequencing according to the manufacturer's instruction to identify the interacting protein partner(s). The AD-T-antigen and BD-p53 interaction was considered as positive control.
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