Y2H assays were performed using the Matchmaker Gold Yeast Two‐Hybrid System (Clontech, CA) according to the manufacturer's instructions. The N‐terminal 190 amino acid residues of GhWRKY41 (GhWRKY41ΔC) were inserted into pGBKT7 and introduced into the yeast strain Y2HGold to generate BD‐GhWRKY41ΔC bait. The primers used are listed in Table S4 .The library that we used for this experiment was a cotton cDNA library prepared from cotton roots under infection conditions with V. dahliae using Match‐maker Library Construction and Screening Kits (Clontech, CA). To confirm the interaction between GhWRKY41 and itself, the full‐length of GhWRKY41 was inserted into pGADT7 to generate GhWRKY41‐AD and introduced into yeast strain Y187. The positive clones of the bait and prey were mixed and mated in 30 °C shaker for 24 h, followed cultured on SD‐Leu‐Trp, SD‐Leu‐Trp‐His (with X‐α‐Gal, Coolaber, Beijing, China) and SD‐Leu‐Trp‐His‐Ade (with X‐α‐Gal) medium.
X α gal
Manufactured by Coolaber
Sourced in China
X-α-Gal is a chromogenic substrate used in microbiology for the detection and identification of bacteria that produce the enzyme α-galactosidase. When the substrate is cleaved by the enzyme, it produces a blue-colored product, enabling the visual detection of positive results.
Automatically generated - may contain errors
Lab products found in correlation
Yeastmaker yeast transformation system 2, by Takara Bio (2 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (2 mentions)
Ppr3n vector, by Takara Bio (1 mentions)
Matchmaker library construction and screening kit, by Takara Bio (1 mentions)
Matchmaker gold yeast two hybrid system, by Takara Bio (1 mentions)
Sd trp, by Coolaber (1 mentions)
Sd leu trp, by Coolaber (1 mentions)
Seamless cloning and assembly kit, by Novoprotein (1 mentions)
X α gal, by Takara Bio (1 mentions)
6 protocols using x α gal
1
GhWRKY41 Self-Interaction Assay
2
Yeast Two-Hybrid Screening of Soybean Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Homologous recombination was used to design specific primers for full-length PCR amplification (Table S1 ). The full-length coding sequences (CDSs), S domain (1–16aa) P domain (17–135 aa) and C domain (136–305 aa) of the Gm1-MMP were cloned into pBT3SUC vector (Clontech, Shanghai, China) according to the different domain functions, respectively. Additionally, the full-length coding sequences (CDSs), N domain (1–34aa) and C domain (35–85 aa) of the GmMT-II were cloned into pPR3N vector (Clontech, Shanghai, China) (Table S1 ). The constructed positive plasmids were used for the co-transformation of into yeast strain AH109 containing the His3 and LacZ reporter genes (Clontech, Shanghai, China), after which the protein–protein interactions were examined in selective medium lacking Trp, Leu, His, and Ade and supplemented with the optimal x-α-gal (Coolaber, Beijing, China).
3
Yeast Two-Hybrid Assay for MAPK Pathway Components
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CDS regions of fus3, ste7, ste11, and ste11 were amplified from total cDNA, purified with a Gel Pure Kit columns I (Magen), and attached to linearized pGADT7-AD and pGBKT7-BD with a ClonExpress II One Step Cloning Kit (Vazyme Biotech Co.). The constructed plasmids were sequenced in Sangon Biotech (Shanghai, China), extracted with a TIANprep mini Plasmid Kit (Transgene, Beijing, China), then pairwise co-transfected into the Y2HGold cells by using a Yeastmaker Yeast Transformation System 2 (630439, TaKaRa). All of the selective medium and reagents, including SD/-Leu, SD/-Trp, SD/-Leu/-Trp, SD/-His/-leu/-Trp/-His, and X-α-gal, were purchased from Coolaber (Beijing, China). All primers used in this experiment are listed in Table S5.
4
Yeast Two-Hybrid Assay for PqMYBF1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The target vector pGBKT7-PqMYBF1 was constructed using a seamless cloning and assembly kit (Novoprotein, Shanghai, China), and the primer sequences are shown in Table S4 . The PGBKT7-PqMYBF1 vector and a negative control of pGBKT7 vector only containing the BD domain of GAL protein were introduced into yeast Y2H competent cells, respectively. Monoclonal yeast plaques of pGBKT7-PqMYBF1 and pGBKT7 plasmids were picked and dissolved in 200 µL 0.09% Nacl solution and adjusted to be OD600 = 1. The absorbed dilutions were incubated on SD/-Trp, SD/-Trp + 40 µg/mL X-α-Gal (Coolaber, Beijing, China), SD/-Trp + 40µg/mL X-α-Gal + 200 ng/mL AbA (Clontech, Mountain View, CA, USA) medium at 29 °C for about 72 h to observe the formation of plaque and coloration.
5
Yeast Two-Hybrid Screening of Mi2G02
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A cDNA library was constructed by extracting RNA from A. thaliana roots infected with M. incognita (1, 3, 5, 10, and 14 dpi) and used to screen for the target proteins of Mi2G02. The Y2H assay was performed according to the Clontech protocol (Clontech, USA). Interactions between Mi2G02 and candidate proteins were assessed in pairwise Y2H assays. Relative BD and AD vectors were used to transform the yeast strain Y2HGold for screening on SD/-Leu-Trp plates. Positive clones were verified and selected for culture on SD/-Leu-Trp-His medium supplemented with 20 mg/ml X-α-Gal (Coolaber, cat. no. SL0940). We investigated whether the nuclear localization sequences of Mi2G02 were required for interaction by inserting a mutated Mi2G02 in the BD vector for pairwise Y2H assays.
The Y1H assay was performed as described previously (Kong et al., 2023 (link)). The sequenced pB42AD and pLacZi vectors were integrated into the yeast strain EGY48 grown on SD/-Trp-Ura medium (Coolaber, cat. no. PM2262). Positive transformants were verified and selected for growth on medium containing raffinose (Coolaber, cat. no. SL0990) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal; Coolaber, cat. no. CX11921) for color reactions.
The Y1H assay was performed as described previously (Kong et al., 2023 (link)). The sequenced pB42AD and pLacZi vectors were integrated into the yeast strain EGY48 grown on SD/-Trp-Ura medium (Coolaber, cat. no. PM2262). Positive transformants were verified and selected for growth on medium containing raffinose (Coolaber, cat. no. SL0990) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal; Coolaber, cat. no. CX11921) for color reactions.
6
Yeast Two-Hybrid Assay for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CDS regions of ssnF, rcoA, and creA were amplified via total cDNA. Using the ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China), the CDS fragments were inserted into pGBKT7 and pGADT7, respectively. The constructed plasmids were sequenced in Sangon Biotech (Figure S4 ), then, pairwise, co-transformed into the Y2HGold cell by the Yeastmaker™ Yeast Transformation System 2 (630439, Takara, Dalian, China). All of the selective media, including SD (lacking leucine)/-Leu, SD (lacking tryptophan)/-Trp, SD (lacking leucine and tryptophan)/-Leu/-Trp, SD (lacking histidine, leucine, tryptophan, and adenine)/-His/-leu/-Trp/-Ade and X-α-gal were purchased from Coolaber (Beijing, China). The Y2H Gold cells containing pGBKT7-p53 and pGADT7-T were set as the positive control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!