The yeast two-hybrid assays were performed as previously described (Wan et al., 2015 (link)). Briefly, Rtt107NTD, as well as various fragments of Nse6, Slx4, Mms22, Scm3, Rad55, and Cdc7 was cloned into pBTM116 (BD) and pACT2 (AD) vectors (Clontech) at the BamHI and XhoI sites (KEY RESOURCES TABLE ). Mutations were made by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). Yeast cells harboring BD and AD plasmids were selected on SC-Leu-Trp plates. β-galactosidase activities were measured according to Clontech Matchmaker yeast two-hybrid protocol with o-nitrophenyl β-D-galactopyranoside (ONPG) as substrate. The averages from three transformants were calculated and error bars represent standard deviations.
Pbtm116
Manufactured by Takara Bio
Sourced in France
The PBTM116 is a laboratory equipment product from Takara Bio. It functions as a thermal cycler, primarily used for DNA amplification through the polymerase chain reaction (PCR) process. The device is capable of precisely controlling temperature and duration for the various stages of the PCR procedure.
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7 protocols using pbtm116
1
Yeast Two-Hybrid Assay for Protein-Protein Interactions
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Yeast Two-Hybrid Screening for RPGRIP1L Interactors
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YTH screen was performed in Saccharomyces cerevisiae strain L40 (trp1-901, his3D200, leu2–3, ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lac GAL4) using MyoVa-GTD cloned into pBTM116 (LexA DNA-binding domain, DBD) as bait and a human fetal brain cDNA library (Clontech) cloned into pACT2 (Gal4 activation domain, AD) as prey. Yeast cells were transformed with pBTM116_MyoVa-GTD vector and the library as described by Alborghetti and co-workers47 (link). The screen was performed in solid Synthetic Defined Medium without tryptophan, leucine and histidine (SD-WLH) containing 5 mM 3-amino-1,2,4-triazole (3-AT) (Sigma-Aldrich, St. Louis, MO). To identify the preys, the pACT2 plasmids of positive clones were isolated and sequenced. The DNA sequences were then compared with those available in the NCBI data bank using the BLASTX program48 (link). The clone identified as encoding for the full-length RPGRIP1L isoform c (NP_001295263.1) was further selected for in vitro and in cell validation and characterization.
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Yeast Two-Hybrid Assay Protocol
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The yeast two-hybrid assay was performed as described previously (Xue et al, 2017 (link)). Briefly, the L40 strain was transformed with pBTM116 and pACT2 (Clontech) fusion plasmids, and colonies harboring both plasmids were selected on Yeast complete–Leu–Trp plates. The β-galactosidase activities were measured by a liquid assay.
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Yeast Two-Hybrid Protein Interaction Assay
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The yeast two-hybrid analyses of the protein-protein interactions were performed using either the Gal4-based plasmid system (pGAD-C1, pGBD-C1 [James et al., 1996 (link)]) in PJ69-7a cells, or the lexA-based plasmid system (pBTM116, Clontech Saint-Germain-en-Laye, France) in L40 cells (Invitrogen Schwerte, Germany).
Transformants were spotted in serial (1:5) or single dilution either on SC-Leu-Trp plates (control) or on SC-Leu-Trp-His plates (selection) and grown at 30°C for 2–4 days. For a specific interaction between TOPBP1 1–360 and SMARCAD1 55–247, spotting plates were supplemented with different concentrations of 3-Amino-1,2,4-triazole (3-AT) (2.5–10 mM). To assess the phosphorylation-specific interaction between SMARCAD1 and Dpb11, cells were additionally spotted on SC-Leu-Trp-His-Ade plates.
All experiments (Figure 1A,G and H ; Figure 9A,C,E ) were performed in three technical repetitions per biological repetition (spotting of the same yeast cultures on three separate selection plates) and each interaction was observed in several (2-10) independent experiments (a biological replicate corresponds to a fresh transformation of the Y2H expression vectors, raising of the transformed cells and spotting on selective plates).
Transformants were spotted in serial (1:5) or single dilution either on SC-Leu-Trp plates (control) or on SC-Leu-Trp-His plates (selection) and grown at 30°C for 2–4 days. For a specific interaction between TOPBP1 1–360 and SMARCAD1 55–247, spotting plates were supplemented with different concentrations of 3-Amino-1,2,4-triazole (3-AT) (2.5–10 mM). To assess the phosphorylation-specific interaction between SMARCAD1 and Dpb11, cells were additionally spotted on SC-Leu-Trp-His-Ade plates.
All experiments (
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Yeast Two-Hybrid Assay for Protein Interactions
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The yeast two-hybrid assay was performed as described previously [58 (link)]. Briefly, the L40 strain was transformed with pBTM116 and pACT2 (Clontech) fusion plasmids, and colonies harboring both plasmids were selected on YC (Yeast complete)–Leu–Trp plates. The β-Galactosidase activities were measured by a liquid assay.
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Yeast Two-Hybrid Assay for SLX4 Interactions
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Wild-type and mutant human SLX4 cDNA fragments were inserted into the modified yeast two-hybrid assay vectors pBTM116 and pACT2 (Clontech), respectively. Yeast two-hybrid constructs were co-transformed into the yeast strain L40. Cells were cultured on selective SD–Trp–Leu plates. For liquid β-galactosidase assay, yeast cells were grown in the SD–Trp–Leu liquid media, permeabilized by three cycles of freeze-and-thaw treatment, and the β-galactosidase activity was determined by measuring OD420 using ortho-Nitrophenyl-β-galactoside (ONPG) as the substrate. All readings were normalized to the density of yeast cells (OD600). All experiments were performed with three independent replicates.
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Yeast two-hybrid assay protocol
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