The cDNA sequences of PIAL1, PIAL2, MOM2, MORC6, and MOM1 CMM2 domain (aa1660-aa1860)5 (link) were first cloned into gateway entry vectors followed by LR reaction with pGBKT7-GW (Addgene 61703) and pGADT7-GW (Addgene 61702) destination vectors. Pairs of plasmid DNA for the desired protein interaction to be tested were co-transformed into the yeast strain AH109. Combinations of the empty pGBKT7-GW or pGADT7-GW vectors and the plasmids of desired proteins were used for transformation of yeast cells to test for self-activation. Transformed yeast cells were plated on synthetic dropout medium without Trp and Leu (SD-TL) and incubated for 2-3 days to allow for the growth of positive colonies carrying both plasmids. Three yeast colonies of each tested protein interaction pairs were picked and mixed in 150μl 1xTE solution, and 3μl of the 1xTE solution with the yeast cells were blotted on synthetic dropout medium without Trp, Leu, and His (SD-TLH) and with 5mM 3-amino-1,2,4-triazole (3AT) to inhibit background growth. Growth of yeast on SD-TLH with 5mM 3AT medium after 2-3 days of incubation indicates the interaction between the GAL4-AD fusion protein and the GAL4-BD fusion protein.
Pgbkt7 gw
Manufactured by Addgene
The PGBKT7-GW is a plasmid vector that can be used for Gateway cloning. It contains a kanamycin resistance gene for selection and the Gateway cassette for efficient cloning of DNA sequences.
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5 protocols using pgbkt7 gw
1
Yeast Two-Hybrid Protein Interaction Assay
2
Cloning CDK8 and Mad for Yeast Two-Hybrid
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Full-length CDK8 was amplified from a pBS-CDK8 cDNA clone using primers CDK8-5.1 (F: 5’-caccATGGACTACG ATTTCAAGAT-3’) and CDK8-3.1 (F: 5’-TCAGTTGAAGCGCTGGAAGT-3’), and then inserted into the pENTR/D-TOPO vector. The Gateway LR Clonase II Enzyme mix was used to recombine CDK8 cDNA into the pGADT7-GW (prey) vector, a gift from Yuhai Cui (Addgene plasmid # 61702) [96 (link)]. The linker region of Mad was amplified with Mad-5.2 and Mad-3.2 primers from a cDNA clone of the Mad gene (LD12679) using PrimeStar Max premix and inserted into the pENTR/D-TOPO vector. All pENTR Mad fragments were recombined into the pGBKT7-GW (bait) vector, a gift from Yuhai Cui (Addgene plasmid # 61703) [96 (link)], using the Gateway LR Clonase II Enzyme mix. The Y2H assay was performed using the AH109 yeast strain, as described previously [96 (link)].
3
Validating Protein-Protein Interactions Using Yeast Two-Hybrid
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To validate the predicted protein–protein interaction between two of our candidate TFs, AP2 and WRKY16, we used the GAL4 yeast two-hybrid system (Clontech). AP2 and WRKY16 were cloned into pGADT7-GW (Addgene Plasmid #61702) and pGBKT7-GW (Addgene Plasmid #61703) plasmids (Lu et al., 2010 (link)). Empty pGADT7 (with the GAL4 activation domain, AD) and pGBKT7 (with the GAL4 DNA-binding domain, BD) plasmids were used as negative controls. We use the yeast (Saccharomyces cerevisiae) strain AH109 in the MATCHMAKER GAL4 Two-Hybrid System (Clontech), in which HIS3, ADE2, MEL1, and LacZ are under the control of GAL4 TF. The AD and BD plasmids were co-transformed to yeast AH109 competent cells following Clontech’s user manual instructions of the polyethylene glycol/lithium acetate method and cultured in YPD Plus Medium for 90 min to promote transformation efficiency. Transformed yeast cells were assayed by culturing in SD/–Leu/–Trp medium to select for successful co-transformants and then assayed by culturing in SD/–Ade/–His/–Leu/–Trp medium with 40 μg·mL−1 X-α-Gal, which provides high-stringency selection. The positive protein–protein interactions between two TFs are indicated by growth on SD/–Ade/–His/–Leu/–Trp/X-α-Gal medium plates and blue colony color.
4
Yeast Two-Hybrid Analysis of Plant Interactome
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The cDNA sequences of PIAL1, PIAL2, MOM2, MORC6, and MOM1 CMM2 domain (aa1660-aa1860)5 (link) were first cloned into gateway entry vectors followed by LR reaction with pGBKT7-GW (Addgene 61703) and pGADT7-GW (Addgene 61702) destination vectors. Pairs of plasmid DNA for the desired protein interaction to be tested were co-transformed into the yeast strain AH109. Combinations of the empty pGBKT7-GW or pGADT7-GW vectors and the plasmids of desired proteins were used for transformation of yeast cells to test for self-activation. Transformed yeast cells were plated on synthetic dropout medium without Trp and Leu (SD-TL) and incubated for 2–3 days to allow for the growth of positive colonies carrying both plasmids. Three yeast colonies of each tested protein interaction pairs were picked and mixed in 150 μL 1×TE solution, and 3 μL of the 1×TE solution with the yeast cells were blotted on synthetic dropout medium without Trp, Leu, and His (SD-TLH) and with 5 mM 3-amino-1,2,4-triazole (3AT) to inhibit background growth. Growth of yeast on SD-TLH with 5 mM 3AT medium after 2–3 days of incubation indicates the interaction between the GAL4-AD fusion protein and the GAL4-BD fusion protein.
5
Yeast Two-Hybrid Protein Interaction Assay
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To test protein-protein interactions coding sequences of CPT3 and LEW1 were subcloned into the pENTR/D-TOPO vector and next recombined into Y2H vectors (pGADT7-GW and pGBKT7-GW) using LR Clonase II. Selected constructs were transformed into S. cerevisiae AH109 strain [MATa, 112, gal4
Serialdilutionsof theselecteddoubletransf ormantsweregrowninplateslackingleucine, tryptophanandhistidine(-Leu/ T rp/-His)supplementedwith1mM 3-AT (3-Amino-1, 2, 4-triazole).T heexperimentswereperf ormedinatleastthreereplic Y2H vectors pGADT7-GW (Addgene plasmid #61702; http://n2t.net/addgene:61702; RRID:Addgene -61702) and pGBKT7-GW (Addgene plasmid #61703; http://n2t.net/addgene:61703 ; RRID:Addgene -61703) were a gift from Yuhai Cui (Lu et al., 2010) .
Serialdilutionsof theselecteddoubletransf ormantsweregrowninplateslackingleucine, tryptophanandhistidine(-Leu/ T rp/-His)supplementedwith1mM 3-AT (3-Amino-1, 2, 4-triazole).T heexperimentswereperf ormedinatleastthreereplic Y2H vectors pGADT7-GW (Addgene plasmid #61702; http://n2t.net/addgene:61702; RRID:Addgene -61702) and pGBKT7-GW (Addgene plasmid #61703; http://n2t.net/addgene:61703 ; RRID:Addgene -61703) were a gift from Yuhai Cui (Lu et al., 2010) .
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