top10 competent cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher top10 competent cells
    Expression and purification of recombinant TbINO1 in E. coli . A. TbINO1 was cloned into the expression vector pBAD TA (C-terminal hexa-His tag) and transformed into <t>TOP10</t> E. coli competent cells, and production of recombinant TbINO1 was induced with 0.2% arabinose. After cell disruption, the soluble TbINO1 was purified by affinity chromatography using a chelating column charged with Ni 2+ , and proteins were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, total E. coli cellular protein after induction; lane 2, soluble E. coli protein after induction, which was loaded onto affinity chromatography column; lane 3, purified recombinant TbINO1 after affinity chromatography; lane 4, purified recombinant TbINO1 after dialysis. B. Kinetics of recombinant TbINO1 for glucose 6-phosphate. Enzyme activity was measured as described in Experimental procedures , NAD + concentration was held constant (1 mM) and glucose 6-phosphate concentration varied. Insert shows Lineweaver-Burk plot of data.
    Top10 Competent Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/top10 competent cells/product/Thermo Fisher
    Average 99 stars, based on 166 article reviews
    Price from $9.99 to $1999.99
    top10 competent cells - by Bioz Stars, 2020-05
    99/100 stars

    Images

    1) Product Images from "The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol"

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2006.05216.x

    Expression and purification of recombinant TbINO1 in E. coli . A. TbINO1 was cloned into the expression vector pBAD TA (C-terminal hexa-His tag) and transformed into TOP10 E. coli competent cells, and production of recombinant TbINO1 was induced with 0.2% arabinose. After cell disruption, the soluble TbINO1 was purified by affinity chromatography using a chelating column charged with Ni 2+ , and proteins were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, total E. coli cellular protein after induction; lane 2, soluble E. coli protein after induction, which was loaded onto affinity chromatography column; lane 3, purified recombinant TbINO1 after affinity chromatography; lane 4, purified recombinant TbINO1 after dialysis. B. Kinetics of recombinant TbINO1 for glucose 6-phosphate. Enzyme activity was measured as described in Experimental procedures , NAD + concentration was held constant (1 mM) and glucose 6-phosphate concentration varied. Insert shows Lineweaver-Burk plot of data.
    Figure Legend Snippet: Expression and purification of recombinant TbINO1 in E. coli . A. TbINO1 was cloned into the expression vector pBAD TA (C-terminal hexa-His tag) and transformed into TOP10 E. coli competent cells, and production of recombinant TbINO1 was induced with 0.2% arabinose. After cell disruption, the soluble TbINO1 was purified by affinity chromatography using a chelating column charged with Ni 2+ , and proteins were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, total E. coli cellular protein after induction; lane 2, soluble E. coli protein after induction, which was loaded onto affinity chromatography column; lane 3, purified recombinant TbINO1 after affinity chromatography; lane 4, purified recombinant TbINO1 after dialysis. B. Kinetics of recombinant TbINO1 for glucose 6-phosphate. Enzyme activity was measured as described in Experimental procedures , NAD + concentration was held constant (1 mM) and glucose 6-phosphate concentration varied. Insert shows Lineweaver-Burk plot of data.

    Techniques Used: Expressing, Purification, Recombinant, Clone Assay, Plasmid Preparation, Transformation Assay, Affinity Chromatography, SDS Page, Staining, Affinity Column, Activity Assay, Concentration Assay

    Related Articles

    Clone Assay:

    Article Title: Crystallographic Insights into the Pore Structures and Mechanisms of the EutL and EutM Shell Proteins of the Ethanolamine-Utilizing Microcompartment of Escherichia coli ▿ ▿ †
    Article Snippet: .. The resulting products were cloned into a pET101 vector (Invitrogen) according to the company's protocol and were subsequently transformed into chemically competent TOP10 cells (Invitrogen). .. Plasmids of clones that were positively identified by PCR were transformed into BL21-AI cells for overexpression.

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

    Amplification:

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

    Ligation:

    Article Title: Generation of Human PTH1R Construct with Flag Epitope Located Internally: Comparison of Two-Fragment Assembly by Using PCR Overlap Extension or Ligase
    Article Snippet: .. The ligation mixture was then transformed to competent cells (Invitrogen) and screened by ampicillin. ..

    Construct:

    Article Title: The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae
    Article Snippet: .. The pLES94 construct was transformed into One Shot TOP10 chemically competent E. coli cells (Invitrogen) by heat shock, and transformants were selected on LB agar containing 100 μg/ml ampicillin and 40 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) and screened by PCR and sequencing. .. A confirmed pLES94- lptA plasmid was purified by use of a miniprep kit (Qiagen) and transformed into strain FA19 to generate strain FA19::PlptA-lacZ .

    Purification:

    Article Title: Alternative Spermidine Biosynthetic Route Is Critical for Growth of Campylobacter jejuni and Is the Dominant Polyamine Pathway in Human Gut Microbiota *
    Article Snippet: .. The product was first purified using QIAquick® PCR purification kit (Qiagen) according to the manufacturer's instructions, before being ligated into pGEM®-T Easy and transformed into E. coli TOP10 competent cells (Invitrogen). .. Clones (white on X-Gal LB plates) were checked by sequencing using M13 universal forward and reverse primers and correct inserts retained.

    Polymerase Chain Reaction:

    Article Title: The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae
    Article Snippet: .. The pLES94 construct was transformed into One Shot TOP10 chemically competent E. coli cells (Invitrogen) by heat shock, and transformants were selected on LB agar containing 100 μg/ml ampicillin and 40 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) and screened by PCR and sequencing. .. A confirmed pLES94- lptA plasmid was purified by use of a miniprep kit (Qiagen) and transformed into strain FA19 to generate strain FA19::PlptA-lacZ .

    Article Title: Alternative Spermidine Biosynthetic Route Is Critical for Growth of Campylobacter jejuni and Is the Dominant Polyamine Pathway in Human Gut Microbiota *
    Article Snippet: .. The product was first purified using QIAquick® PCR purification kit (Qiagen) according to the manufacturer's instructions, before being ligated into pGEM®-T Easy and transformed into E. coli TOP10 competent cells (Invitrogen). .. Clones (white on X-Gal LB plates) were checked by sequencing using M13 universal forward and reverse primers and correct inserts retained.

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

    Incubation:

    Article Title: What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities
    Article Snippet: .. Vectors (4µl) were transformed into Escherichia coli cells by incubation with 25µl competent E. coli cells (DH5α; Invitrogen, Renfrewshire, UK) at 4°C for 30 minutes, followed by a heatshock of 42°C for 45 seconds and rotary incubation with 475µl Super Optimal broth with Catabolite repression (SOC) at 37°C for 1 hour. ..

    Sequencing:

    Article Title: The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae
    Article Snippet: .. The pLES94 construct was transformed into One Shot TOP10 chemically competent E. coli cells (Invitrogen) by heat shock, and transformants were selected on LB agar containing 100 μg/ml ampicillin and 40 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) and screened by PCR and sequencing. .. A confirmed pLES94- lptA plasmid was purified by use of a miniprep kit (Qiagen) and transformed into strain FA19 to generate strain FA19::PlptA-lacZ .

    Transformation Assay:

    Article Title: The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae
    Article Snippet: .. The pLES94 construct was transformed into One Shot TOP10 chemically competent E. coli cells (Invitrogen) by heat shock, and transformants were selected on LB agar containing 100 μg/ml ampicillin and 40 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) and screened by PCR and sequencing. .. A confirmed pLES94- lptA plasmid was purified by use of a miniprep kit (Qiagen) and transformed into strain FA19 to generate strain FA19::PlptA-lacZ .

    Article Title: Generation of Human PTH1R Construct with Flag Epitope Located Internally: Comparison of Two-Fragment Assembly by Using PCR Overlap Extension or Ligase
    Article Snippet: .. The ligation mixture was then transformed to competent cells (Invitrogen) and screened by ampicillin. ..

    Article Title: Genetic Analysis of the Regulation of Type IV Pilus Function by the Chp Chemosensory System of Pseudomonas aeruginosa ▿
    Article Snippet: .. Chemically competent E. coli cells (Invitrogen) were transformed according to the manufacturer's instructions. .. E. coli and P. aeruginosa liquid cultures were maintained in Luria-Bertani (LB) broth, and solid medium was prepared by adding 0.8 to 1.5% agar (Bacto agar; Becton Dickinson).

    Article Title: Engineering a Virus-Like Particle as an Antigenic Platform for a Pfs47-Targeted Malaria Transmission-Blocking Vaccine
    Article Snippet: .. Briefly, BL21(DE3) pLysS chemically competent E. coli cells (ThermoFisher) or E. coli OverExpress™ C41(DE3) (Lucigen) were transformed with pEt17b-AP205-SpyCatcher or pET24-AP205-SpyCatcher and inoculated onto LB agar plates with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol. .. Ten transformed BL21 colonies were picked and cultured in 10 separate tubes containing 4 mL LB supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol O/N at 37 °C.

    Article Title: Crystallographic Insights into the Pore Structures and Mechanisms of the EutL and EutM Shell Proteins of the Ethanolamine-Utilizing Microcompartment of Escherichia coli ▿ ▿ †
    Article Snippet: .. The resulting products were cloned into a pET101 vector (Invitrogen) according to the company's protocol and were subsequently transformed into chemically competent TOP10 cells (Invitrogen). .. Plasmids of clones that were positively identified by PCR were transformed into BL21-AI cells for overexpression.

    Article Title: Alternative Spermidine Biosynthetic Route Is Critical for Growth of Campylobacter jejuni and Is the Dominant Polyamine Pathway in Human Gut Microbiota *
    Article Snippet: .. The product was first purified using QIAquick® PCR purification kit (Qiagen) according to the manufacturer's instructions, before being ligated into pGEM®-T Easy and transformed into E. coli TOP10 competent cells (Invitrogen). .. Clones (white on X-Gal LB plates) were checked by sequencing using M13 universal forward and reverse primers and correct inserts retained.

    Article Title: What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities
    Article Snippet: .. Vectors (4µl) were transformed into Escherichia coli cells by incubation with 25µl competent E. coli cells (DH5α; Invitrogen, Renfrewshire, UK) at 4°C for 30 minutes, followed by a heatshock of 42°C for 45 seconds and rotary incubation with 475µl Super Optimal broth with Catabolite repression (SOC) at 37°C for 1 hour. ..

    Plasmid Preparation:

    Article Title: Crystallographic Insights into the Pore Structures and Mechanisms of the EutL and EutM Shell Proteins of the Ethanolamine-Utilizing Microcompartment of Escherichia coli ▿ ▿ †
    Article Snippet: .. The resulting products were cloned into a pET101 vector (Invitrogen) according to the company's protocol and were subsequently transformed into chemically competent TOP10 cells (Invitrogen). .. Plasmids of clones that were positively identified by PCR were transformed into BL21-AI cells for overexpression.

    Article Title: The olfactory secretome varies according to season in female sheep and goat
    Article Snippet: .. PCR products were cloned into pCR4®-TOPO vector (TOPO™-TA cloning™ kit, Invitrogen), then amplified into Escherichia coli One Shot™ Top10 chemically competent cells (Invitrogen). .. Recombinant plasmids were purified with QIAprep Spin Miniprep kit (Qiagen) and sequenced in both senses (Eurofins Genomics).

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