Sd leu trp medium

Manufactured by Takara Bio

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SD/-Leu/-Trp medium is a selective growth medium used for the cultivation of yeast cells that have been transformed with plasmids containing the LEU2 and TRP1 genes. This medium lacks the amino acids leucine and tryptophan, requiring the transformed yeast cells to rely on the plasmid-encoded genes for growth.

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Lab products found in correlation

Sd trp leu his ade medium, by Takara Bio (2 mentions) X α gal, by Takara Bio (2 mentions) Yeast protocols handbook, by Takara Bio (1 mentions) Phis2, by Takara Bio (1 mentions) Y2hgold yeast, by Takara Bio (1 mentions) Matchmaker y2h system, by Takara Bio (1 mentions) Matchmaker gold y2h system, by Takara Bio (1 mentions) Aureobasidin a, by Takara Bio (1 mentions)

Spelling variants of this product

SD/-Leu/-Trp (26 citations) SD/−Trp (7 citations) SD-Trp-Leu-His-Ade medium (6 citations) SD medium (3 citations)

8 protocols using sd leu trp medium

Yeast Two-Hybrid Screening for Protein Interactions

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For validating the protein interactions, prey and bait vectors containing genes as indicated in the figures were co‐transformed into Y‐2‐HGold chemically competent cells. Positive yeast clones containing two plasmids were selected from SD/‐Trp‐Leu medium (#630494; Clontech, Terra Bella Avenue Mountain View, CA, USA) and were re‐plated on SD/‐Trp‐Leu and SD/‐Trp‐Leu‐His‐Ade medium (#630494; Clontech).
After codon optimization and synthesis, the CDSs of ScPEPR1‐N and SsPE14 were ligated into plasmids, generating pCzn1_ScPEPR1‐N and pGEX‐4T‐1_SsPE14, which were transformed into Escherichia coli strain BL21. The histidine (HIS)‐ and GST‐tagged proteins were purified and the in vitro GST pulldown experiments were performed according to the method of Tarun & Sachs (1996 (link)).

Ling H., Fu X., Huang N., Zhong Z., Su W., Lin W., Cui H, & Que Y. (2021). A sugarcane smut fungus effector simulates the host endogenous elicitor peptide to suppress plant immunity. The New Phytologist, 233(2), 919-933.

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Yeast Two-Hybrid Screening of NPR1 and EIN3

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The pGADT7 and pGBKT7 vectors were used, and polyethylene glycolinduced transformations were performed according to the instructions in the Yeast Protocols Handbook (Clontech). An N-terminal fragment of the NPR1 coding sequence (1 to 194 amino acids) and a fragment lacking the N-terminal region (178 to 593 amino acids) were separately cloned into the pGBKT7 vector. Similarly, EIN3 coding sequence fragments (1 to 500 amino acids and full length) were inserted into the pGADT7 vector. Plasmid pairs were cotransformed into AH109 cells that were incubated at 30°C on selective dropout (SD)-Trp-Leu medium (Clontech). Finally, the AH109 cells were selected on SD medium lacking Trp, Leu, His, and adenine (Ade; SD-Trp-Leu-His-Ade).

Huang P., Dong Z., Guo P., Zhang X., Qiu Y., Li B., Wang Y, & Guo H. (2020). Salicylic Acid Suppresses Apical Hook Formation via NPR1-Mediated Repression of EIN3 and EIL1 in Arabidopsis. The Plant cell, 32(3).

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Yeast Two-Hybrid Interaction Assay

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The coding sequences of MaAPX1 and MaMsrB2 were subcloned into pGBKT7-BD or pGADT7-AD vectors to create bait and prey constructs. Then, the paired DNA-binding domain (BD) and transcription-activating domain (AD) constructs were co-transformed into the yeast strain AH109 (BioMed) using the lithium acetate method. Yeast cells were grown on minimal synthetic defined-double-dropouts (SD-Leu/-Trp) medium (Clontech) for 3 days. The co-transformants were then transferred onto quadruple dropout (QDO) (SD-Leu/-Trp/-Ade/-His) medium to test the possible interactions based on their growth status. The ability of yeast cells to grow on QDO medium was scored as a positive interaction.

Xiao L., Jiang G., Yan H., Lai H., Su X., Jiang Y, & Duan X. (2021). Methionine Sulfoxide Reductase B Regulates the Activity of Ascorbate Peroxidase of Banana Fruit. Antioxidants, 10(2), 310.

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Yeast Two-Hybrid System for Bait-Prey Interactions

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The 43-bp NWRE fragment amplified by PCR was inserted into pHIS2.1 (Clontech, CA, USA) to generate the plasmid pHIS2.1-NWRE as bait. The coding regions of IbbHLH3 and IbbHLH4 were separately cloned into pGADT7 as effectors. Lithium acetate-mediated transformation [53 (link)] was performed to transform the constructs into yeast strain Y187. In a preliminary nutrient deficiency screening, the transformants were grown on SD/-Leu/-Trp medium (Clontech) at 30°C for 4 days. The surviving transformants were further selected using SD/-Leu/-Trp/-His medium with 100 mM 3-AT (3-amino-1,2,4-triazol; Sigma, USA) at 30°C for 4 days.

Chen S.P., Kuo C.H., Lu H.H., Lo H.S, & Yeh K.W. (2016). The Sweet Potato NAC-Domain Transcription Factor IbNAC1 Is Dynamically Coordinated by the Activator IbbHLH3 and the Repressor IbbHLH4 to Reprogram the Defense Mechanism against Wounding. PLoS Genetics, 12(10), e1006397.

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Yeast Two-Hybrid Assay for Protein Interactions

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To detect protein interactions, plasmids were transformed into Y2H-Gold Yeast (Clontech) cells using a lithium acetate and polyethylene glycolmediated protocol. After incubation for 2 d at 28°C in synthetic defined (SD)/-Leu-Trp medium (Clontech), the yeast clones were transferred to SD/-Leu-Trp-His-Ade medium (Clontech) with or without chemical treatment. To assay the effects of rac-GR24, GR24 5DS , GR24 ent-5DS , and KAR 1 on the interaction of OsSMAX1 and D14L, the yeasts cotransformed with OsSMAX1-binding domain and D14L-AD were diluted to different concentrations and grown for 60 h on SD/-Leu-Trp-His-Ade medium with the indicated chemicals at 20 mM. rac-GR24 and KAR 1 were obtained from Chiralix (Nijmegen), and the GR24 stereoisomers GR24 5DS and GR24 ent- 5DS were prepared as described by Wang et al. (2020) .

Zheng J., Hong K., Zeng L., Wang L., Kang S., Qu M., Dai J., Zou L., Zhu L., Tang Z., Meng X., Wang B., Hu J., Zeng D., Zhao Y., Cui P., Wang Q., Qian Q., Wang Y., Li J, & Xiong G. (2020). Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness. The Plant cell, 32(9).

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Evaluating Protein Interactions in Wheat

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Protein interactions among TaSYP137 and TaVAMP723 were evaluated using the MatchMaker Y2H system (Clontech Inc. Palo Alto, USA), as previously described [63 (link)]. The full-length TaSYP137 and TaVAMP723 ORF was amplified by PCR with specific primers with a homologous arm designed using an NCBI primer blast and ligated into the PGBKT7 plasmid (Table S2). The transcriptional activity of the transformants was evaluated by preparing ten-fold serial dilutions and using 3 μL aliquots to inoculate SD/-Leu-Trp-Ade-His medium and SD/-Leu-Trp medium containing X-α-Gal (Clontech Inc. Palo Alto, USA). The inoculated media were incubated at 30 °C for 4 days. The coding sequences were then inserted into the BD and AD vectors.

Wang G., Zhang X., Guo H., Zhao C., Zhang H., Chen C, & Ji W. (2023). TaSYP137 and TaVAMP723, the SNAREs Proteins from Wheat, Reduce Resistance to Blumeria graminis f. sp. tritici. International Journal of Molecular Sciences, 24(5), 4830.

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Yeast Two-Hybrid Screening Protocol

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Y2H assays were performed using the Matchmaker Gold Y2H System following the manufacturer’s protocol (Clontech, Mountain View, CA, USA). Briefly, coding sequences of the target genes were amplified with gene-specific primers (see Table S3) and cloned into pGBKT7 or pGADT7 vectors. The constructed vectors were cotransformed into yeast strain Y2H Gold using the lithium acetate/single-stranded DNA/PEG method (96 (link)). The pair of pGBKT7-53 and pGADT7-T was used as a positive control. Cotransformed yeast cells were grown on SD/-Leu/-Trp medium (Clontech, Mountain View, CA, USA) at 30°C for 5 days. The transformants were further screened on QDO medium (Clontech, Mountain View, CA, USA) containing 40 μg mL⁻¹ X-α-Gal (Clontech, Mountain View, CA, USA) or 125 ng mL⁻¹ aureobasidin A (Clontech, Mountain View, CA, USA).

Gao Y., Xiong X., Wang H., Bi Y., Wang J., Yan Y., Li D, & Song F. (2023). Fusarium oxysporum f. sp. niveum Pumilio 1 Regulates Virulence on Watermelon through Interacting with the ARP2/3 Complex and Binding to an A-Rich Motif in the 3′ UTR of Diverse Transcripts. mBio, 14(2), e00157-23.

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Yeast Two-Hybrid Assay of STN1 and TEN1

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The MATCHMAKER two-hybrid system 3 (http://www.bdbiosciences.com) was used for this experiment, whereby STN1 was used as the bait and TEN1 with or without the G100E mutation was used as the prey. The STN1 and TEN1 coding sequences were amplified by PCR using cDNA made from wild-type Col seedlings as the template, cut with EcoRI along with PstI and BamHI, respectively, and then cloned into the corresponding sites of the GBKT7 or GADT7 vectors. After confirmation by Sanger sequencing, the STN1-BD and TEN1-AD or TEN1-G100E-AD plasmids, as well as negative controls, were cotransformed into competent yeast (Saccharomyces cerevisiae) cells (strain: YH109), and cells with both constructs were selected on SD/-Leu/-Trp medium (Clontech, 630417). Ten colonies picked up with a tooth pick were diluted in 1 mL 0.9% NaCl (w/v) and spotted onto SD/-Ade/-His/-Leu/-Trp medium (Clontech, 630428) with or without α-X-Gal (GoldBio, 107021-38-5). See Supplemental Table S3 for information about the primers.

Wang B., Shi X., Gao J., Liao R., Fu J., Bai J, & Cui H. (2023). SCARECROW maintains the stem cell niche in Arabidopsis roots by ensuring telomere integrity. Plant physiology, 192(2).

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