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ii one step cloning kit  (Vazyme Biotech Co)


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    Vazyme Biotech Co ii one step cloning kit
    Ii One Step Cloning Kit, supplied by Vazyme Biotech Co, used in various techniques. Bioz Stars score: 99/100, based on 6257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/one+step+cloning+kit/ClonExpress+II+One+Step+Cloning+Kit/pmc13090716-105-13-18
    Average 99 stars, based on 6257 article reviews
    ii one step cloning kit - by Bioz Stars, 2026-09
    99/100 stars

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    Cloning:

    Article Title: Mutation of lysine 396 of the fusion protein reduces its binding affinity with integrin αVβ1 and leads to attenuation of avian metapneumovirus subtype B.
    Article Snippet: .. Following the instructions of the ClonExpress II one-step cloning kit (C112, Vazyme), the 12 linearized vectors and gene fragments were recombined to obtain 12 full-length cDNA infectious clone plasmids with the following gene substitutions: pOKLN16-VAN, pOKLN16-VAP, pOKLN16-VAF, pOKLN16-VASH, pOKLN16-VAG, pOKLN16VAL, pOKLN16-AVN, pOKLN16-AVP, pOKLN16-AVF, pOKLN16-AVSH, pOKLN16-AVG, and pOKLN16-AVL, respectively. .. Subsequently, using pOKLN16-VAF and pOKLN16-AVF as templates, amino acids 323, 396, and 522 of the F protein were sequentially mutated using the Mut Express II rapid mutagenesis kit (C214, Vazyme).

    Article Title: Metabolic engineering and adaptive laboratory evolution enhance squalene production in Yarrowia lipolytica
    Article Snippet: The fragments were obtained by PCR and recovered using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). .. Primer's information is provided in Plasmid construction was carried out using the ClonExpress II One-Step Cloning Kit (Vazyme, China), and plasmid extraction was performed with the AxyPrep Plasmid Miniprep Kit (Axygen, USA). ..

    Article Title: Enhanced production and anti-inflammatory activity of exopolysaccharides through the overexpression of β-1,3-glucanosyltransferase gene in Ganoderma lingzhi.
    Article Snippet: This study aimed to enhance the production and anti-inflammatory activity of exopolysaccharides (EPS) in Ganoderma lingzhi through genetic engineering.. The β-1,3-glucanosyltransferase gene of the glycosyl-hydrolase 72 family was cloned and overexpressed in G. lingzhi.. The overexpressed strain exhibited a maximum intracellular polysaccharide content of 26.49 ± 0.19 mg/100 mg dry cell weight and an EPS production of 1.16 ± 0.22 g/L, which were 28% and 23% higher, respectively, than those of the wild-type (WT) strain.

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Article Title: Stepwise engineering of AmpR-based whole-cell biosensors for broad-spectrum detection and high-throughput screening of β-lactam compounds.
    Article Snippet: Cephalosporin C (CPC), a representative secondary metabolite of filamentous fungi, serves as the precursor for the industrial synthesis of 7-aminocephalosporanic acid (7-ACA), the key intermediate for semisynthetic cephalosporins.. Metabolite-oriented high-throughput screening is essential for accelerating the engineering of β-lactam-producing fungi for enhanced production and of key enzymes within β-lactam biosynthetic pathways for desirable activity.. To create a biosensor compatible with droplet-based microfluidics, we systematically engineered an AmpR-based fluorescent Escherichia coli whole-cell biosensor and obtained the superior variant AmpRR86H/L199S, which exhibits a 23.9-fold dynamic range for CPC, a lowered limit of detection, and broad specificity toward four major β-lactam classes and 7-ACA.

    Article Title: Developing Emericellopsis sp. XJ1056 as a versatile fungal platform for high-yield biosynthesis of nonribosomal peptides: a case study on beauvericin
    Article Snippet: PCR amplifications were performed using 2× Phanta Max Master Mix (Vazyme Biotech, Nanjing, China). .. DNA fragments were assembled using the ClonExpress MultiS one-step cloning kit (Vazyme Biotech, Nanjing, China). .. PCR products were purified with the Axygen ® DNA Gel Extraction Kit (Corning, NY, United States).

    Article Title: Block-building with yeast to elucidate an artificial pathway for de novo biosynthesis of glabridin.
    Article Snippet: To facilitate seamless gene insertion via Gibson assembly, gene fragments (either synthesized or PCR-amplified) were digested with DpnI (Takara bio). .. The assembly reactions were performed using ClonExpress Ultra One-Step Cloning Kit (Vazyme, Nanjing, China). ..

    Plasmid Preparation:

    Article Title: Metabolic engineering and adaptive laboratory evolution enhance squalene production in Yarrowia lipolytica
    Article Snippet: The fragments were obtained by PCR and recovered using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). .. Primer's information is provided in Plasmid construction was carried out using the ClonExpress II One-Step Cloning Kit (Vazyme, China), and plasmid extraction was performed with the AxyPrep Plasmid Miniprep Kit (Axygen, USA). ..

    Article Title: Enhanced production and anti-inflammatory activity of exopolysaccharides through the overexpression of β-1,3-glucanosyltransferase gene in Ganoderma lingzhi.
    Article Snippet: This study aimed to enhance the production and anti-inflammatory activity of exopolysaccharides (EPS) in Ganoderma lingzhi through genetic engineering.. The β-1,3-glucanosyltransferase gene of the glycosyl-hydrolase 72 family was cloned and overexpressed in G. lingzhi.. The overexpressed strain exhibited a maximum intracellular polysaccharide content of 26.49 ± 0.19 mg/100 mg dry cell weight and an EPS production of 1.16 ± 0.22 g/L, which were 28% and 23% higher, respectively, than those of the wild-type (WT) strain.

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Article Title: Stepwise engineering of AmpR-based whole-cell biosensors for broad-spectrum detection and high-throughput screening of β-lactam compounds.
    Article Snippet: Cephalosporin C (CPC), a representative secondary metabolite of filamentous fungi, serves as the precursor for the industrial synthesis of 7-aminocephalosporanic acid (7-ACA), the key intermediate for semisynthetic cephalosporins.. Metabolite-oriented high-throughput screening is essential for accelerating the engineering of β-lactam-producing fungi for enhanced production and of key enzymes within β-lactam biosynthetic pathways for desirable activity.. To create a biosensor compatible with droplet-based microfluidics, we systematically engineered an AmpR-based fluorescent Escherichia coli whole-cell biosensor and obtained the superior variant AmpRR86H/L199S, which exhibits a 23.9-fold dynamic range for CPC, a lowered limit of detection, and broad specificity toward four major β-lactam classes and 7-ACA.

    Extraction:

    Article Title: Metabolic engineering and adaptive laboratory evolution enhance squalene production in Yarrowia lipolytica
    Article Snippet: The fragments were obtained by PCR and recovered using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). .. Primer's information is provided in Plasmid construction was carried out using the ClonExpress II One-Step Cloning Kit (Vazyme, China), and plasmid extraction was performed with the AxyPrep Plasmid Miniprep Kit (Axygen, USA). ..

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Amplification:

    Article Title: Enhanced production and anti-inflammatory activity of exopolysaccharides through the overexpression of β-1,3-glucanosyltransferase gene in Ganoderma lingzhi.
    Article Snippet: This study aimed to enhance the production and anti-inflammatory activity of exopolysaccharides (EPS) in Ganoderma lingzhi through genetic engineering.. The β-1,3-glucanosyltransferase gene of the glycosyl-hydrolase 72 family was cloned and overexpressed in G. lingzhi.. The overexpressed strain exhibited a maximum intracellular polysaccharide content of 26.49 ± 0.19 mg/100 mg dry cell weight and an EPS production of 1.16 ± 0.22 g/L, which were 28% and 23% higher, respectively, than those of the wild-type (WT) strain.

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Article Title: Stepwise engineering of AmpR-based whole-cell biosensors for broad-spectrum detection and high-throughput screening of β-lactam compounds.
    Article Snippet: Cephalosporin C (CPC), a representative secondary metabolite of filamentous fungi, serves as the precursor for the industrial synthesis of 7-aminocephalosporanic acid (7-ACA), the key intermediate for semisynthetic cephalosporins.. Metabolite-oriented high-throughput screening is essential for accelerating the engineering of β-lactam-producing fungi for enhanced production and of key enzymes within β-lactam biosynthetic pathways for desirable activity.. To create a biosensor compatible with droplet-based microfluidics, we systematically engineered an AmpR-based fluorescent Escherichia coli whole-cell biosensor and obtained the superior variant AmpRR86H/L199S, which exhibits a 23.9-fold dynamic range for CPC, a lowered limit of detection, and broad specificity toward four major β-lactam classes and 7-ACA.

    Polymerase Chain Reaction:

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Article Title: Stepwise engineering of AmpR-based whole-cell biosensors for broad-spectrum detection and high-throughput screening of β-lactam compounds.
    Article Snippet: Cephalosporin C (CPC), a representative secondary metabolite of filamentous fungi, serves as the precursor for the industrial synthesis of 7-aminocephalosporanic acid (7-ACA), the key intermediate for semisynthetic cephalosporins.. Metabolite-oriented high-throughput screening is essential for accelerating the engineering of β-lactam-producing fungi for enhanced production and of key enzymes within β-lactam biosynthetic pathways for desirable activity.. To create a biosensor compatible with droplet-based microfluidics, we systematically engineered an AmpR-based fluorescent Escherichia coli whole-cell biosensor and obtained the superior variant AmpRR86H/L199S, which exhibits a 23.9-fold dynamic range for CPC, a lowered limit of detection, and broad specificity toward four major β-lactam classes and 7-ACA.

    Ligation:

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Transformation Assay:

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Incubation:

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Clone Assay:

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction. .. Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.

    Transfection:

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).

    Pull Down Assay:

    Article Title: Osteoblastic sclerostin loop3-LRP4 interaction required by sclerostin to inhibit bone formation.
    Article Snippet: .. After ligating the PCR product, the Flag-tagged DNA fragments and linear carrier with the one-step cloning kit (Vazyme, C112-01), further the ligation products were transformed into E.coli DH5α competent cells and incubated on the solid agar plates more than 12 h. Colony PCR and restriction enzyme digestion were used to pick up positive clones for subsequent plasmid amplification and extraction.36 Transfections were performed in the MC3T3-E1 cells with Lipomaster 3000 transfection Reagent (Vazyme, TL301) according to the manufacturer’s instructions.37 Pull-down assay. .. Cell lysates were collected using RIPA Lysis and Extraction Buffer (Thermo Scientific, 89901) supplemented with Protease Inhibitor Cocktail (CST, 5871) and centrifuged at 4 000 r/ min for 30 min at 4 °C and replaced with binding buffer (PBS buffer, 20 mmol/L imidazole, pH 8.0).



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