top10 escherichia coli  (Thermo Fisher)


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    Structured Review

    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer"

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.13183

    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Figure Legend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Techniques Used: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    2) Product Images from "Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer"

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.13183

    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Figure Legend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Techniques Used: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    Related Articles

    Methylation Sequencing:

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Paragraph title: TA cloning and bisulfite sequencing ... Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Clone Assay:

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
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    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
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    Article Title: Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP
    Article Snippet: The cDNA was excised from pRSET with Bam HIand Nhe I, which cut in the plasmid's multiple cloning region 5' to the insert and in the cDNA's 3'-untranslated region, respectively. .. The pTrxFus and pThioHis constructs were used to transform GI724 and GI698, and Top10 E. coli respectively (InVitrogen) [ ].

    Article Title: Design and construction of generalizable RNA-protein hybrid controllers by level-matched genetic signal amplification
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    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
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    Amplification:

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    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
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    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: TA cloning and bisulfite sequencing Bisulfite‐converted DNA was amplified by polymerase chain reaction (PCR) using Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Fremont, CA, USA) in a T100 Thermal Cycler (Bio‐Rad). .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: .. The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen). .. The sequence identity was confirmed, and the sequence was cloned into the expression vector pAM 104-2 and transformed into the Rosetta DE3TM E. coli (Novagen) expression system.

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: Cloning of the CD46 isoforms BC1, BC2, C1, and C2 The open reading frame (ORF) of each of the CD46 isoforms, BC1, BC2, C1, and C2 was amplified by PCR from the HCT-116 cell line or PBMCs using DreamTaq DNA polymerase (Thermo Scientific, USA) and 500 nM of the following primers (TAG Copenhagen, Denmark); fw: 5′-ATGGAGCCTCCCGGCCG, rev-13: 5′-GTCAGAGAGAAGTAAATTTTACTTCTCTGTGGGTC (for amplifying BC1 and C1), and rev-14: 5′-GTCAGCCTCTCTGCTCTGCTGG (for amplifying the BC2 and C2). .. The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions.

    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
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    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
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    Random Hexamer Labeling:

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    Synthesized:

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    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
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    TA Cloning:

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
    Article Snippet: The amplified fragment was cloned into pcDNA3.3 (Invitrogen, Karlsruhe, Germany) using the Topo TA Cloning system (Invitrogen), and pcDNA3.3- ST6GALNAC5 was created. .. The vector was amplified in TOP10 E. coli (Invitrogen) and site directed mutagenesis on the recovered plasmid was performed using the QuickChange site-directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) to create plasmid pcDNA3.3- ST6GALNAC5 - p.Val99Met carrying the c.G295A mutation (NM_030965.1).

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Paragraph title: TA cloning and bisulfite sequencing ... Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Construct:

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
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    Article Title: Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP
    Article Snippet: .. The pTrxFus and pThioHis constructs were used to transform GI724 and GI698, and Top10 E. coli respectively (InVitrogen) [ ]. .. The reading frames of all plasmid constructs were confirmed by DNA sequencing.

    Real-time Polymerase Chain Reaction:

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: Primer pairs for quantitative PCR analysis were tested on four brachial and four femoral artery cDNAs using iQ SYBR Green Supermix reagents on an iCycler Real-Time PCR Detection System (Bio-Rad Inc., Hercules, CA, USA). .. PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA).

    Incubation:

    Article Title: Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Article Snippet: The mixture was incubated in a PCR machine at 50°C for 1 h, with the hot-lid set at 105°C. .. 2 µl of the assembly mixture was then transformed into TOP10 E. coli (Invitrogen).

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Activity Assay:

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
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    Expressing:

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
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    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
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    Article Title: Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Article Snippet: .. Growth and induction For all expression experiments in , vectors or isothermal assembly reactions were transformed into TOP10 E. coli (Invitrogen) using either the manufacturer's instructions or the TSS competent cell method as previously described ( ). .. Individual colonies were inoculated into 1 ml of LB + 1.0% glucose + 50 µg/ml ampicillin, and grown overnight at 30°C in a 2 ml deep-well plate (Thermo Scientific) with shaking at 1200 rpm on a Titramax 1000 platform shaker (Heidolph).

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
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    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
    Article Snippet: Paragraph title: Expression of CHIPS in E. coli. ... The vector was transformed into TOP10 E. coli and recombinant CHIPS was expressed and purified according to the manufacturer's instructions (Invitrogen).

    Article Title: A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia
    Article Snippet: For heterologous expression in Saccharomyces cerevisiae (yeast) the coding sequence was introduced into the yeast expression vector pYES-DEST52 (Thermo Fisher Scientific) using LR Clonase II (Thermo Fisher Scientific). .. The resulting PCR products were digested with Dpn1 and transformed into TOP10 Escherichia coli (Thermo Fisher Scientific).

    Transformation Assay:

    Article Title: Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Article Snippet: .. 2 µl of the assembly mixture was then transformed into TOP10 E. coli (Invitrogen). .. Growth and induction For all expression experiments in , vectors or isothermal assembly reactions were transformed into TOP10 E. coli (Invitrogen) using either the manufacturer's instructions or the TSS competent cell method as previously described ( ).

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: .. The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA). ..

    Article Title: Design and construction of generalizable RNA-protein hybrid controllers by level-matched genetic signal amplification
    Article Snippet: .. All cloned plasmid candidates were transformed into TOP10 Escherichia coli (Thermo Fisher Scientific), and plated on LB agar plates (EMD Millipore) with ampicillin (50 mg/L) for overnight growth in 37°C. .. The next day single colonies on the plates were first PCR screened with primers that bind 200~500 base pairs upstream and downstream of the inserts and further verified by sequencing.

    Article Title: Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Article Snippet: .. Growth and induction For all expression experiments in , vectors or isothermal assembly reactions were transformed into TOP10 E. coli (Invitrogen) using either the manufacturer's instructions or the TSS competent cell method as previously described ( ). .. Individual colonies were inoculated into 1 ml of LB + 1.0% glucose + 50 µg/ml ampicillin, and grown overnight at 30°C in a 2 ml deep-well plate (Thermo Scientific) with shaking at 1200 rpm on a Titramax 1000 platform shaker (Heidolph).

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen). .. The sequence identity was confirmed, and the sequence was cloned into the expression vector pAM 104-2 and transformed into the Rosetta DE3TM E. coli (Novagen) expression system.

    Article Title: Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    Article Snippet: .. The MTP5 RT-PCR product was cloned into the pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA). .. Sequence analysis of the TOPO-MTP5 plasmid showed that the MTP5 sequence was mis-spliced (5′ of the mtp5-1 mutation) compared to annotation by The Arabidopsis Information Resource (TAIR; www.arabidopsis.org ).

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: .. PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA). ..

    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
    Article Snippet: .. The vector was transformed into TOP10 E. coli and recombinant CHIPS was expressed and purified according to the manufacturer's instructions (Invitrogen). ..

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia
    Article Snippet: .. The resulting PCR products were digested with Dpn1 and transformed into TOP10 Escherichia coli (Thermo Fisher Scientific). .. All mutations were verified by Sanger sequencing.

    High Performance Liquid Chromatography:

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen). .. Generated NC4 domain of collagen IX were extracted from bacteria under native conditions ( ) and purified by Ni2+ affinity using a HisTrapTM HP column attached to an ÄKTA high-performance liquid chromatography system as described above.

    Ligation:

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: The 3′deoxyadenosine overhang generated by the Taq polymerase was used for ligation of the PCR products into the pcDNA3.1/V5-His-TOPO vector (Invitrogen, USA). .. The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions.

    Cell Culture:

    Article Title: Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
    Article Snippet: Yeast strains were cultured in yeast extract peptone dextrose (YPD) or synthetic complete media (SC) with appropriate amino acids dropped out. .. Cloning was performed in Top10 Escherichia coli grown in either Luria Broth (LB) or Super Optimal broth with Catabolite repression (SOC) media (Invitrogen, Cat. 15544-034).

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Generated:

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: .. The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen). .. The sequence identity was confirmed, and the sequence was cloned into the expression vector pAM 104-2 and transformed into the Rosetta DE3TM E. coli (Novagen) expression system.

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: The 3′deoxyadenosine overhang generated by the Taq polymerase was used for ligation of the PCR products into the pcDNA3.1/V5-His-TOPO vector (Invitrogen, USA). .. The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions.

    Polymerase Chain Reaction:

    Article Title: Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Article Snippet: The mixture was incubated in a PCR machine at 50°C for 1 h, with the hot-lid set at 105°C. .. 2 µl of the assembly mixture was then transformed into TOP10 E. coli (Invitrogen).

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: PCR amplification was undertaken for 35 cycles with the following conditions: denaturation at 95°C for 15 sec., annealing at 60°C for 30 sec. and elongation at 72°C for 60 sec. .. The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
    Article Snippet: Creation of ST6GALNAC5 –containing vectors and measurement of sialyltransferase enzyme activity ST6GALNAC5 cDNA was PCR amplified from a human heart Multiple_Tissue_cDNA_Panel (Clontech, Heidelberg, Germany); the reverse primer was designed to encode the FLAG-tag. .. The vector was amplified in TOP10 E. coli (Invitrogen) and site directed mutagenesis on the recovered plasmid was performed using the QuickChange site-directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) to create plasmid pcDNA3.3- ST6GALNAC5 - p.Val99Met carrying the c.G295A mutation (NM_030965.1).

    Article Title: Design and construction of generalizable RNA-protein hybrid controllers by level-matched genetic signal amplification
    Article Snippet: Pfu Hotstart polymerase or PfuUltra II (Agilent Technologies) was used for PCR reactions for DNA fragments shorter or longer than 2 kb, respectively. .. All cloned plasmid candidates were transformed into TOP10 Escherichia coli (Thermo Fisher Scientific), and plated on LB agar plates (EMD Millipore) with ampicillin (50 mg/L) for overnight growth in 37°C.

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: .. The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen). .. The sequence identity was confirmed, and the sequence was cloned into the expression vector pAM 104-2 and transformed into the Rosetta DE3TM E. coli (Novagen) expression system.

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: The 3′deoxyadenosine overhang generated by the Taq polymerase was used for ligation of the PCR products into the pcDNA3.1/V5-His-TOPO vector (Invitrogen, USA). .. The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions.

    Article Title: Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    Article Snippet: The resulting cDNA was PCR-amplified using Pfu Turbo DNA polymerase (Stratagene) and the oligonucleotides MTPc2-4 and MTPc2-3 (5′-T CGGA T CC TCGACGAAGTTGGAACTTTAAGATC-3′). .. The MTP5 RT-PCR product was cloned into the pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA).

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: .. PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA). ..

    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
    Article Snippet: The PCR product was cloned into the pTrcHISB vector (Invitrogen) directly downstream the enterokinase cleavage site. .. The vector was transformed into TOP10 E. coli and recombinant CHIPS was expressed and purified according to the manufacturer's instructions (Invitrogen).

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia
    Article Snippet: .. The resulting PCR products were digested with Dpn1 and transformed into TOP10 Escherichia coli (Thermo Fisher Scientific). .. All mutations were verified by Sanger sequencing.

    CpG Methylation Assay:

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Plasmid DNA was extracted using PureLink Quick Plasmid Miniprep Kit (ThermoFisher) and Sanger sequenced using T3 sequencing primer (Supporting information, Table ) to establish CpG methylation profile on a single cell level.

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Plasmid DNA was extracted using PureLink Quick Plasmid Miniprep Kit (ThermoFisher) and Sanger sequenced using T3 sequencing primer (Supporting information, Table ) to establish CpG methylation profile on a single cell level.

    DNA Sequencing:

    Article Title: Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP
    Article Snippet: The pTrxFus and pThioHis constructs were used to transform GI724 and GI698, and Top10 E. coli respectively (InVitrogen) [ ]. .. The reading frames of all plasmid constructs were confirmed by DNA sequencing.

    Sequencing:

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: The MMP cleavage site prediction tool ( http://www.dmbr.ugent.be/prx/bioit2-public/SitePrediction/index.php ) was used to identify the MMP2 cleavage site at the beginning of the NC1 domain in the collagen IV α5 aa sequence, and primers that recognized the corresponding gene sequence were designed. .. The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
    Article Snippet: The vector was amplified in TOP10 E. coli (Invitrogen) and site directed mutagenesis on the recovered plasmid was performed using the QuickChange site-directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) to create plasmid pcDNA3.3- ST6GALNAC5 - p.Val99Met carrying the c.G295A mutation (NM_030965.1). .. ST6GALNAC5 sequences in the three constructs were verified by Sanger sequencing.

    Article Title: Design and construction of generalizable RNA-protein hybrid controllers by level-matched genetic signal amplification
    Article Snippet: All cloned plasmid candidates were transformed into TOP10 Escherichia coli (Thermo Fisher Scientific), and plated on LB agar plates (EMD Millipore) with ampicillin (50 mg/L) for overnight growth in 37°C. .. The next day single colonies on the plates were first PCR screened with primers that bind 200~500 base pairs upstream and downstream of the inserts and further verified by sequencing.

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Plasmid DNA was extracted using PureLink Quick Plasmid Miniprep Kit (ThermoFisher) and Sanger sequenced using T3 sequencing primer (Supporting information, Table ) to establish CpG methylation profile on a single cell level.

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: A vector containing an E. coli codon bias optimized cDNA sequence coding for the NC4 domain of collagen IX was used as template to amplify the sequence with the primer forward 5′-CACCTCTGCGGCGGTGAAACGT-3′ and reverse 5′-CCTTATTACTGGCTCGGGGTAA-3′. .. The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen).

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions. .. The orientation and correct sequence of the ORFs were verified by Sanger sequencing (GATC, Germany) using the following primers (TAG Copenhagen, Denmark) covering the insert: T7 fw 5′-TAATACGACTCACTATAGGG, and BGH rev: 5′-TAGAAGGCACAGTCGAGG.

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: Quantitative RT-PCR Primer 3 was used to design primers for quantitative PCR from the most representative public ID sequence specified by Affymetrix annotation [ ]. .. PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA).

    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
    Article Snippet: The chp gene, except for the signal sequence, was amplified by PCR on chromosomal DNA of S. aureus Newman using Pwo DNA polymerase (Roche Diagnostics). .. The vector was transformed into TOP10 E. coli and recombinant CHIPS was expressed and purified according to the manufacturer's instructions (Invitrogen).

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Plasmid DNA was extracted using PureLink Quick Plasmid Miniprep Kit (ThermoFisher) and Sanger sequenced using T3 sequencing primer (Supporting information, Table ) to establish CpG methylation profile on a single cell level.

    Article Title: A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia
    Article Snippet: For heterologous expression in Saccharomyces cerevisiae (yeast) the coding sequence was introduced into the yeast expression vector pYES-DEST52 (Thermo Fisher Scientific) using LR Clonase II (Thermo Fisher Scientific). .. The resulting PCR products were digested with Dpn1 and transformed into TOP10 Escherichia coli (Thermo Fisher Scientific).

    Sonication:

    Article Title: Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP
    Article Snippet: The pTrxFus and pThioHis constructs were used to transform GI724 and GI698, and Top10 E. coli respectively (InVitrogen) [ ]. .. The thioredoxin fusion proteins were released from the E. coli by subjecting one ml of a 5.0 OD550 culture resuspended in 20 mM Tris pH 8.0 2.5 mM EDTA to repeated sonication and flash freezing in liquid N2 [ ].

    Recombinant:

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: Paragraph title: Gene cloning, expression and purification of recombinant protein ... The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
    Article Snippet: PcDNA3.3 allows constitutive expression of recombinant genes under the cytomegalovirus promoter. .. The vector was amplified in TOP10 E. coli (Invitrogen) and site directed mutagenesis on the recovered plasmid was performed using the QuickChange site-directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) to create plasmid pcDNA3.3- ST6GALNAC5 - p.Val99Met carrying the c.G295A mutation (NM_030965.1).

    Article Title: Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP
    Article Snippet: The pTrxFus and pThioHis constructs were used to transform GI724 and GI698, and Top10 E. coli respectively (InVitrogen) [ ]. .. Pilot expression cultures confirmed the size of the recombinant protein and were used to optimize the temperature and duration of protein expression.

    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
    Article Snippet: .. The vector was transformed into TOP10 E. coli and recombinant CHIPS was expressed and purified according to the manufacturer's instructions (Invitrogen). ..

    Mutagenesis:

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
    Article Snippet: .. The vector was amplified in TOP10 E. coli (Invitrogen) and site directed mutagenesis on the recovered plasmid was performed using the QuickChange site-directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) to create plasmid pcDNA3.3- ST6GALNAC5 - p.Val99Met carrying the c.G295A mutation (NM_030965.1). .. Overlap extension PCR was performed to create plasmid pcDNA3.3- ST6GALNAC5 - p.*337Qext*20 carrying the c.T1009C mutation.

    Article Title: Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    Article Snippet: The MTP5 RT-PCR product was cloned into the pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA). .. Sequence analysis of the TOPO-MTP5 plasmid showed that the MTP5 sequence was mis-spliced (5′ of the mtp5-1 mutation) compared to annotation by The Arabidopsis Information Resource (TAIR; www.arabidopsis.org ).

    Article Title: A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia
    Article Snippet: Paragraph title: Cloning procedures and mutagenesis ... The resulting PCR products were digested with Dpn1 and transformed into TOP10 Escherichia coli (Thermo Fisher Scientific).

    Isolation:

    Article Title: Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    Article Snippet: Paragraph title: MTP5 cDNA isolation ... The MTP5 RT-PCR product was cloned into the pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA).

    Subcloning:

    Article Title: Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    Article Snippet: Underlined base pairs in MTPc2-3 and MTPc2-4 were altered to create a Sal I or Not I restriction site, respectively, for subsequent subcloning. .. The MTP5 RT-PCR product was cloned into the pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA).

    Size-exclusion Chromatography:

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: PCR amplification was undertaken for 35 cycles with the following conditions: denaturation at 95°C for 15 sec., annealing at 60°C for 30 sec. and elongation at 72°C for 60 sec. .. The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

    Purification:

    Article Title: Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Article Snippet: Paragraph title: Digestion, purification and assembly of part and destination vectors ... 2 µl of the assembly mixture was then transformed into TOP10 E. coli (Invitrogen).

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: Paragraph title: Gene cloning, expression and purification of recombinant protein ... The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: Design and construction of generalizable RNA-protein hybrid controllers by level-matched genetic signal amplification
    Article Snippet: All cloned plasmid candidates were transformed into TOP10 Escherichia coli (Thermo Fisher Scientific), and plated on LB agar plates (EMD Millipore) with ampicillin (50 mg/L) for overnight growth in 37°C. .. Sequencing to verify each candidate colony was performed by Elim Biopharmaceuticals Inc, by sending either purified plasmids or bacterial colonies.

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: Paragraph title: Expression and Purification of the NC4 Domain of Human Collagen Type IX in E. coli ... The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen).

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: The PCR products were purified from a 1% agarose gel (1g w/v agarose (Invitrogen, USA) dissolved in TAE (Tris-NaOH + 2% acetic acid + 1 mM EDTA) (Substrate department, AU, Denmark)). .. The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions.

    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
    Article Snippet: .. The vector was transformed into TOP10 E. coli and recombinant CHIPS was expressed and purified according to the manufacturer's instructions (Invitrogen). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    Article Snippet: .. The MTP5 RT-PCR product was cloned into the pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA). .. Sequence analysis of the TOPO-MTP5 plasmid showed that the MTP5 sequence was mis-spliced (5′ of the mtp5-1 mutation) compared to annotation by The Arabidopsis Information Resource (TAIR; www.arabidopsis.org ).

    Quantitative RT-PCR:

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: Paragraph title: Quantitative RT-PCR ... PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA).

    Plasmid Preparation:

    Article Title: Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly
    Article Snippet: 100 ng of digested PCR-purified destination vector and equimolar amounts of gel-purified parts were combined in a 5 µl volume, and 5 µl of a 2× isothermal assembly aliquot added. .. 2 µl of the assembly mixture was then transformed into TOP10 E. coli (Invitrogen).

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: .. The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA). ..

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
    Article Snippet: .. The vector was amplified in TOP10 E. coli (Invitrogen) and site directed mutagenesis on the recovered plasmid was performed using the QuickChange site-directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) to create plasmid pcDNA3.3- ST6GALNAC5 - p.Val99Met carrying the c.G295A mutation (NM_030965.1). .. Overlap extension PCR was performed to create plasmid pcDNA3.3- ST6GALNAC5 - p.*337Qext*20 carrying the c.T1009C mutation.

    Article Title: Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle - structure/function study of Xenopus IRBP
    Article Snippet: The cDNA was excised from pRSET with Bam HIand Nhe I, which cut in the plasmid's multiple cloning region 5' to the insert and in the cDNA's 3'-untranslated region, respectively. .. The pTrxFus and pThioHis constructs were used to transform GI724 and GI698, and Top10 E. coli respectively (InVitrogen) [ ].

    Article Title: Design and construction of generalizable RNA-protein hybrid controllers by level-matched genetic signal amplification
    Article Snippet: .. All cloned plasmid candidates were transformed into TOP10 Escherichia coli (Thermo Fisher Scientific), and plated on LB agar plates (EMD Millipore) with ampicillin (50 mg/L) for overnight growth in 37°C. .. The next day single colonies on the plates were first PCR screened with primers that bind 200~500 base pairs upstream and downstream of the inserts and further verified by sequencing.

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors *
    Article Snippet: .. The generated PCR fragment was ligated into the vector pET200/D-TOPO (Invitrogen) and amplified in TOP10 E. coli (Invitrogen). .. The sequence identity was confirmed, and the sequence was cloned into the expression vector pAM 104-2 and transformed into the Rosetta DE3TM E. coli (Novagen) expression system.

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: The 3′deoxyadenosine overhang generated by the Taq polymerase was used for ligation of the PCR products into the pcDNA3.1/V5-His-TOPO vector (Invitrogen, USA). .. The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions.

    Article Title: Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    Article Snippet: .. The MTP5 RT-PCR product was cloned into the pCR4Blunt-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA). .. Sequence analysis of the TOPO-MTP5 plasmid showed that the MTP5 sequence was mis-spliced (5′ of the mtp5-1 mutation) compared to annotation by The Arabidopsis Information Resource (TAIR; www.arabidopsis.org ).

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: .. PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA). ..

    Article Title: Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent
    Article Snippet: .. The vector was transformed into TOP10 E. coli and recombinant CHIPS was expressed and purified according to the manufacturer's instructions (Invitrogen). ..

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol. .. Transformed cells were cultured on lysogeny broth (LB) plates with 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) and incubated overnight at 37o C. The following day, 20 colonies were picked from each plate and cultured overnight in LB medium containing 50 μg/ml Kanamycin (Substrate Unit, Karolinska University Hospital) at 37o C at 210 rpm.

    Article Title: A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia
    Article Snippet: For heterologous expression in Saccharomyces cerevisiae (yeast) the coding sequence was introduced into the yeast expression vector pYES-DEST52 (Thermo Fisher Scientific) using LR Clonase II (Thermo Fisher Scientific). .. The resulting PCR products were digested with Dpn1 and transformed into TOP10 Escherichia coli (Thermo Fisher Scientific).

    SYBR Green Assay:

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: Primer pairs for quantitative PCR analysis were tested on four brachial and four femoral artery cDNAs using iQ SYBR Green Supermix reagents on an iCycler Real-Time PCR Detection System (Bio-Rad Inc., Hercules, CA, USA). .. PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: The amplicon (675 bp) was eluted from a 1.5% Agarose gel (Amresco, Cochran Solon, OH, USA) using a QIAEX II gel extraction kit (Qiagen, Doncaster, VIC, Australia) and cloned into pcDNΑ5/FRT/TO-TOPO (Invitrogen) according to the manufacturer's recommendations. .. The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells
    Article Snippet: The PCR products were purified from a 1% agarose gel (1g w/v agarose (Invitrogen, USA) dissolved in TAE (Tris-NaOH + 2% acetic acid + 1 mM EDTA) (Substrate department, AU, Denmark)). .. The plasmids were cloned in TOP10 E. coli (Invitrogen, USA) according to the manufacturer′s instructions.

    Quantitation Assay:

    Article Title: Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine
    Article Snippet: PCR products were cloned into a pCR-4TOPO vector and transformed into TOP10 E. coli (Invitrogen, Inc., Carlsbad, CA, USA). .. Plasmids with sequence-verified inserts were quantified by fluorometry (Picogreen® dsDNA Quantitation Kit, Invitrogen Inc., Carlsbad, CA, USA) for use as quantification standards.

    Concentration Assay:

    Article Title: Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background
    Article Snippet: 5-Fluoroorotic Acid Monohydrate (FOA, 5-FOA) was purchased from US Biological (Cat. F5050) and used at a concentration of 1 mg mL-1 . .. Cloning was performed in Top10 Escherichia coli grown in either Luria Broth (LB) or Super Optimal broth with Catabolite repression (SOC) media (Invitrogen, Cat. 15544-034).

    FLAG-tag:

    Article Title: Mutation in ST6GALNAC5 identified in family with coronary artery disease
    Article Snippet: Creation of ST6GALNAC5 –containing vectors and measurement of sialyltransferase enzyme activity ST6GALNAC5 cDNA was PCR amplified from a human heart Multiple_Tissue_cDNA_Panel (Clontech, Heidelberg, Germany); the reverse primer was designed to encode the FLAG-tag. .. The vector was amplified in TOP10 E. coli (Invitrogen) and site directed mutagenesis on the recovered plasmid was performed using the QuickChange site-directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) to create plasmid pcDNA3.3- ST6GALNAC5 - p.Val99Met carrying the c.G295A mutation (NM_030965.1).

    Gel Extraction:

    Article Title: Lamstatin – a novel inhibitor of lymphangiogenesis derived from collagen IV
    Article Snippet: The amplicon (675 bp) was eluted from a 1.5% Agarose gel (Amresco, Cochran Solon, OH, USA) using a QIAEX II gel extraction kit (Qiagen, Doncaster, VIC, Australia) and cloned into pcDNΑ5/FRT/TO-TOPO (Invitrogen) according to the manufacturer's recommendations. .. The vector was transformed into TOP10 E.coli (Invitrogen) and streaked on agar plates with ampicillin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: PCR amplicons were gel‐purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer
    Article Snippet: PCR amplicons were gel‐purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). .. Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

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    MultiShot StripWell TOP10 chemically competent E coli cells are cloning competent cells that packaged in a rack containing 12 strips of 8 tubes to increase productivity for medium throughput bacterial
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    99
    Thermo Fisher top10 escherichia coli
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into <t>TOP10</t> <t>Escherichia</t> coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Top10 Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher e coli top10 carrying vector pcr4 topo
    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli <t>TOP10</t> cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector <t>pCR4-TOPO,</t> pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.
    E Coli Top10 Carrying Vector Pcr4 Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher escherichia coli
    Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and <t>Escherichia</t> coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.
    Escherichia Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Resistance against sulfonamide antibiotics mediated by SEW2_dhps01, SEW5_dhps01, AEW9_dhps01 , and AEG2_dhps01 . Five microliters of serially diluted E. coli TOP10 cultures with starting OD 600 of 0.5 were spotted onto Iso-Sensitest agar plates supplemented with 1000 mg/L sulfamethazine (+ SMZ), 250 mg/L sulfamethoxazole (+ SMX), 250 mg/L sulfadiazine (+ SDZ) or 500 mg/L sulfisoxazole (+ SOX). Iso-Sensitest agar plates with no sulfonamide added (control) were also included. E. coli TOP10 cultures carrying the cloning vector pCR4-TOPO, pCR4_SEW2_dhps01, pCR4_SEW5_dhps01, pCR4_AEW9_dhps01 or pCR4_AEG2_dhps01 were considered.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Clone Assay, Plasmid Preparation

    Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes

    doi: 10.3389/fmicb.2019.00460

    Figure Lengend Snippet: Antibiotic susceptibility profiles of E. coli TOP10 carrying soil-derived genes involved in antibiotic resistance. The genes were subcloned into plasmid vector pCR4-TOPO. MICs of antibiotics were determined using the broth microdilution method and are presented as fold increase relative to those for E. coli TOP10 carrying the cloning vector pCR4-TOPO. CAX, cefotaxime; CHL, chloramphenicol; ERY, erythromycin; GEN, gentamicin; LIN, lincomycin; RIF, rifampicin; SDZ, sulfadiazine; SMX, sulfamethoxazole; SMZ, sulfamethazine; SOX, sulfisoxazole; TET, tetracycline; TYL, tylosin.

    Article Snippet: E. coli TOP10 carrying vector pCR4-TOPO (Thermo Fisher Scientific) was used as control.

    Techniques: Derivative Assay, Plasmid Preparation, Clone Assay

    Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and Escherichia coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.

    Journal: Journal of dairy science

    Article Title: Immunoproteomics to identify Staphylococcus aureus antigens expressed in bovine milk during mastitis

    doi: 10.3168/jds.2017-14040

    Figure Lengend Snippet: Representative SDS-PAGE, 2-dimensional electrophoresis (2DE), and Western blots showing the workflow for selection of immunoreactive spots. (A) The 1-dimensional SDS-PAGE image of Staphylococcus aureus Newbould 305 trypsinized protein (lane 1) and Escherichia coli trypsinized protein (lane 2). (B) 2DE gel conducted on 7 cm, pH 4 to 7, immobilized pH gradient (IPG) strips of S. aureus Newbould 305 trypsinized protein. Mr = molecular weight. (C) Immunoblot of S. aureus Newbould 305 trypsinized protein using pooled bovine mastitic milk, with immunoreactive regions highlighted in red circles. (D) 2DE gel conducted on 7 cm, pH 4 to 7, IPG strips of Escherichia coli DH10-β trypsinized protein. (E) Immunoblot of E. coli DH10-β trypsinized protein using bovine mastitic milk, with the immunoreactive regions highlighted in red circles. Color version available online.

    Article Snippet: Briefly, S. aureus Newbould 305, S. aureus C1, and Escherichia coli (DH10-β Top10, Thermo Fisher Scientific, Waltham, MA) were grown in LIM overnight to an optical density ( OD ) of 0.75 to 1.2 and harvested by centrifugation (6,000 × g for 10 min at 4°C), before washing 3 times with 1× PBS.

    Techniques: SDS Page, Electrophoresis, Two-Dimensional Gel Electrophoresis, Western Blot, Selection, Molecular Weight