competent top10  (Thermo Fisher)


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    Structured Review

    Thermo Fisher competent top10
    Measured RPU values for promoters assembled in the same plasmid in <t>TOP10</t> and KRX strains. Panels A-D show the measured promoters activities in all the tested conditions. Green and red bars indicate that the promoter has been characterized via GFP32 and RFP34 reporter device respectively. Error bars represent the standard deviation of the mean activity computed on three clones. For each group, the RPU value of the individually characterized promoter is also reported. Statistical analysis was performed via ANOVA test to compare the RPU activities measured in the tested conditions. Promoters showing a statistical difference (P ) in the mean activities among the tested conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P 0.05) are marked with a ‘-’ sign. When a significant difference is present for a given promoter, the statistical analysis was also performed by excluding the conditions where the promoter drives the ‘downstream’ cassette and panel E shows the subset of conditions used for such comparisons, as well as the statistical analysis results. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).
    Competent Top10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/competent top10/product/Thermo Fisher
    Average 90 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    competent top10 - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Bottom-Up Engineering of Biological Systems through Standard Bricks: A Modularity Study on Basic Parts and Devices"

    Article Title: Bottom-Up Engineering of Biological Systems through Standard Bricks: A Modularity Study on Basic Parts and Devices

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039407

    Measured RPU values for promoters assembled in the same plasmid in TOP10 and KRX strains. Panels A-D show the measured promoters activities in all the tested conditions. Green and red bars indicate that the promoter has been characterized via GFP32 and RFP34 reporter device respectively. Error bars represent the standard deviation of the mean activity computed on three clones. For each group, the RPU value of the individually characterized promoter is also reported. Statistical analysis was performed via ANOVA test to compare the RPU activities measured in the tested conditions. Promoters showing a statistical difference (P ) in the mean activities among the tested conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P 0.05) are marked with a ‘-’ sign. When a significant difference is present for a given promoter, the statistical analysis was also performed by excluding the conditions where the promoter drives the ‘downstream’ cassette and panel E shows the subset of conditions used for such comparisons, as well as the statistical analysis results. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).
    Figure Legend Snippet: Measured RPU values for promoters assembled in the same plasmid in TOP10 and KRX strains. Panels A-D show the measured promoters activities in all the tested conditions. Green and red bars indicate that the promoter has been characterized via GFP32 and RFP34 reporter device respectively. Error bars represent the standard deviation of the mean activity computed on three clones. For each group, the RPU value of the individually characterized promoter is also reported. Statistical analysis was performed via ANOVA test to compare the RPU activities measured in the tested conditions. Promoters showing a statistical difference (P ) in the mean activities among the tested conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P 0.05) are marked with a ‘-’ sign. When a significant difference is present for a given promoter, the statistical analysis was also performed by excluding the conditions where the promoter drives the ‘downstream’ cassette and panel E shows the subset of conditions used for such comparisons, as well as the statistical analysis results. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).

    Techniques Used: Plasmid Preparation, Standard Deviation, Activity Assay, Clone Assay

    Related Articles

    Clone Assay:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: The cDNA encoding FpPG was cloned in pGAPZαA (Invitrogen) using the Eco RI and Xba I restriction sites introduced by using the primers EcoFw (5′-acctga gaattc gatccctgctccgtgac-3′) and XbaRv (5′-gccta tctaga ctagctggggcaagtgtt-3′). .. The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells.

    Article Title: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: .. Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA). .. The clone libraries were developed from PCR products generated using fecal and water DNA extracts.

    Article Title: The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem ▿
    Article Snippet: The resultant pRB8535 was digested with XhoI-XbaI, and the Tcr fragment flanked by ICP0 sequence was cloned into pKO5 to generate pKO8535. .. Competent cells of Escherichia coli strain RR1 harboring bacterial accessory chromosome 8502 (BAC8502) ( ) were electroporated with pKO8535 and incubated at 43°C on Lennox LB (Invitrogen, Carlsbad, CA) plates containing zeocin (25 μg/ml) and chloramphenicol (20 μg/ml).

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used. .. HPV clones were amplified by culturing 5-ml amp-LB media overnight at 37°C and re-inoculated in 1 Lt LB media containing 5 mmol/L ampicillin and grown overnight at 37°C.

    Article Title: Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion
    Article Snippet: Paragraph title: Cloning and expression of MBP-linker-MPR-TM and MBP-AAA-MPR-TM ... An aliquot of the LR reaction was used to transform One Shot TOP10 chemically competent E. coli cells following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA).

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Approximately 40 clones per serum were generated and sequenced using the T7 promoter primer ( 5′-CCCTATAGTGAGTCGTATTA-3′ ).

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions. .. Ethics statement All carcass collection, euthanasia, and biopsy sampling procedures were performed under the authority of other agencies in accordance with applicable state laws; specimens were submitted for routine diagnostic analyses that did not require further review by NWHC institutional committees.

    Article Title: Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
    Article Snippet: Briefly, the full-length cDNAs were subcloned into the pENTER/D-TOPO vector using a pENTER Directional TOPO Cloning kit (Invitrogen). .. After purification of the plasmids from the transformed Top10 competent cells (Invitrogen) the cDNA inserts were transferred to the pAd (adenovirus)/CMV/V5-DEST vector (Invitrogen) by means of the Gateway® system using LR Clonase™ (Invitrogen).

    Amplification:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: .. The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells. .. Transformants were selected on low-salt Luria-Bertani plates containing 25 μg/mL zeocin (Duchefa Biochemie) and analyzed by direct PCR amplification using specific primers of the α-factor signal peptide and the 3′-Alcohol Oxidase terminator sequence according to the manufacturer’s instruction.

    Article Title: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: In addition, the class-specific epsilonproteobacterial 23S rRNA gene primers EPS_23S_1325F (5′-TCGTCATCGTAGGGTTAG-3′) and EPS_23S_1763R (5′-GTAAACAGTCGGGAGGGA-3′) were used specifically for the amplification of Campylobacter and Helicobacter against 18 fecal and three water samples ( ). .. Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA).

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: Amplified fragments from HPV-16 were cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and the amplified fragments from HPV-18, HPV-45, and HPV-51 were cloned into the pGEM-T easy vector (Promega, Madison, WI) according to the specifications of the manufacturers. .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used.

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: Sequencing using amplification primers resulted in the characterization of a 10,075 nt sequence. .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen).

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: Obtained amplification products were sequenced with the Sanger method (Seqlab, Germany and Eurofins MWG Biotech, Germany). .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing.

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: The amplified product was purified using a QIAquick PCR purification kit (Qiagen) and ligated into pBAD TOPO TA (Invitrogen) vector and sequence confirmed. .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression.

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: Amplified DNA from 5 randomly selected PCR- and culture-positive samples and all culture-negative samples that produced positive PCR results were sequenced to confirm that the amplicons matched the targeted region. .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions.

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: Fusion PCR was then performed to combine upstream and downstream products with a 660 bp amplicon of the cat gene ( ). .. This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen).

    Binding Assay:

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: This in vitro transposition system places unique primer binding sites randomly in a population of large DNA molecules. .. Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD).

    Synthesized:

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: Fragments C2, C9 and C12 were synthesized using a two-step protocol: cDNA was generated using a mixture of random hexaprimers (RT Taqman Applied Biosystems) followed by PCR amplification using Taq Polymerase (Invitrogen). .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen).

    TA Cloning:

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Approximately 40 clones per serum were generated and sequenced using the T7 promoter primer ( 5′-CCCTATAGTGAGTCGTATTA-3′ ).

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing. .. Alignment and analysis of obtained sequences were carried out using BioEdit, version 7.2.5 ( ).

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions. .. Ethics statement All carcass collection, euthanasia, and biopsy sampling procedures were performed under the authority of other agencies in accordance with applicable state laws; specimens were submitted for routine diagnostic analyses that did not require further review by NWHC institutional committees.

    Construct:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: .. The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells. .. Transformants were selected on low-salt Luria-Bertani plates containing 25 μg/mL zeocin (Duchefa Biochemie) and analyzed by direct PCR amplification using specific primers of the α-factor signal peptide and the 3′-Alcohol Oxidase terminator sequence according to the manufacturer’s instruction.

    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: Paragraph title: Two Fragments of F1 and F2 and Linearized Vector Were Assembled Sequentially by Ligase to Generate Flag-PTH1R Construct ... The ligation mixture was then transformed to competent cells (Invitrogen) and screened by ampicillin.

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression. ..

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: Primers adjacent to the upstream and downstream regions of hylB were constructed with 25 bp 5′ extensions corresponding to the 5′ and 3′ ends of the cat gene from pACYC (see for list of primers) ( ). .. This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen).

    Electrophoresis:

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: The specificity of the amplification was evaluated by agarose-gel electrophoresis and by sequence analysis. .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used.

    Nested PCR:

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: Genetic characterization of the E-gene was conducted using the Qiagen OneStep RT-PCR kit together with primers E1F-E4R, followed by a nested PCR using primers E2F-E3R ( ) to produce a 1,759 nt fragment including the complete E-gene (1,485 nt) which was subsequently sequenced directly using amplification primers. .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen).

    Incubation:

    Article Title: The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem ▿
    Article Snippet: .. Competent cells of Escherichia coli strain RR1 harboring bacterial accessory chromosome 8502 (BAC8502) ( ) were electroporated with pKO8535 and incubated at 43°C on Lennox LB (Invitrogen, Carlsbad, CA) plates containing zeocin (25 μg/ml) and chloramphenicol (20 μg/ml). ..

    Expressing:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: Paragraph title: FpPG Expression and Purification ... The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells.

    Article Title: Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion
    Article Snippet: Paragraph title: Cloning and expression of MBP-linker-MPR-TM and MBP-AAA-MPR-TM ... An aliquot of the LR reaction was used to transform One Shot TOP10 chemically competent E. coli cells following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA).

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression. ..

    Article Title: Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
    Article Snippet: Paragraph title: Adenovirus expression system ... After purification of the plasmids from the transformed Top10 competent cells (Invitrogen) the cDNA inserts were transferred to the pAd (adenovirus)/CMV/V5-DEST vector (Invitrogen) by means of the Gateway® system using LR Clonase™ (Invitrogen).

    Transformation Assay:

    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: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: .. Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA). .. The clone libraries were developed from PCR products generated using fecal and water DNA extracts.

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used. .. HPV clones were amplified by culturing 5-ml amp-LB media overnight at 37°C and re-inoculated in 1 Lt LB media containing 5 mmol/L ampicillin and grown overnight at 37°C.

    Article Title: Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation
    Article Snippet: .. p21 sense plasmid DNA Plasmid DNA was isolated from competent E-coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen, Carlsbad CA) or the vector containing the full-length p21WAF1/CIP1 gene in the sense direction described by us [ ]. .. Mixed Lymphocyte Reaction (MLR) MLR was performed with splenocytes from isografts, untreated and CsA treated transplant recipients (LEW) as responders and irradiated splenocytes from donor strain (WF) as stimulators.

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Approximately 40 clones per serum were generated and sequenced using the T7 promoter primer ( 5′-CCCTATAGTGAGTCGTATTA-3′ ).

    Article Title: Development of novel O-polysaccharide based glycoconjugates for immunization against glanders
    Article Snippet: .. Chemically competent E. coli TOP10 cells were transformed as per the manufacturer's instructions (Invitrogen). .. Oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, IA).

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression. ..

    Article Title: Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
    Article Snippet: .. After purification of the plasmids from the transformed Top10 competent cells (Invitrogen) the cDNA inserts were transferred to the pAd (adenovirus)/CMV/V5-DEST vector (Invitrogen) by means of the Gateway® system using LR Clonase™ (Invitrogen). ..

    Hybridization:

    Article Title: The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem ▿
    Article Snippet: Competent cells of Escherichia coli strain RR1 harboring bacterial accessory chromosome 8502 (BAC8502) ( ) were electroporated with pKO8535 and incubated at 43°C on Lennox LB (Invitrogen, Carlsbad, CA) plates containing zeocin (25 μg/ml) and chloramphenicol (20 μg/ml). .. The colonies grown on the sucrose plates were screened by colony hybridization.

    Electroporation:

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen). .. After its propagation in MC1061 E. coli , the pKO hylB construct was introduced into WT GBS through electroporation.

    Transfection:

    Article Title: Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
    Article Snippet: After purification of the plasmids from the transformed Top10 competent cells (Invitrogen) the cDNA inserts were transferred to the pAd (adenovirus)/CMV/V5-DEST vector (Invitrogen) by means of the Gateway® system using LR Clonase™ (Invitrogen). .. The linearized plasmids (1–2 μg) were then mixed with 4 μl of Lipofectamine™ 2000 (Invitrogen) in 200 μl of Opti-MEM® medium (Invirogen) and transfected into subconfluent 293A cells (Invitrogen) in 1 ml of Opti-MEM® in 6 well plates (Iwaki Glass).

    Sequencing:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: .. The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells. .. Transformants were selected on low-salt Luria-Bertani plates containing 25 μg/mL zeocin (Duchefa Biochemie) and analyzed by direct PCR amplification using specific primers of the α-factor signal peptide and the 3′-Alcohol Oxidase terminator sequence according to the manufacturer’s instruction.

    Article Title: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: Paragraph title: Cloning and sequencing analyses. ... Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA).

    Article Title: The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem ▿
    Article Snippet: The resultant pRB8535 was digested with XhoI-XbaI, and the Tcr fragment flanked by ICP0 sequence was cloned into pKO5 to generate pKO8535. .. Competent cells of Escherichia coli strain RR1 harboring bacterial accessory chromosome 8502 (BAC8502) ( ) were electroporated with pKO8535 and incubated at 43°C on Lennox LB (Invitrogen, Carlsbad, CA) plates containing zeocin (25 μg/ml) and chloramphenicol (20 μg/ml).

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: The specificity of the amplification was evaluated by agarose-gel electrophoresis and by sequence analysis. .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used.

    Article Title: Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion
    Article Snippet: Cloning and expression of MBP-linker-MPR-TM and MBP-AAA-MPR-TM The coding region of gp41 MPR-TM flanked by a tobacco etch virus (TEV) cleavage site was subcloned from pMistic-MPR-TM [ ] into the pCR8/GW/Topo vector (Invitrogen, Carlsbad, CA), sequence-verified and shuttled into the pVP16 destination vector [ ] by Gateway recombination cloning (Invitrogen, Carlsbad, CA). .. An aliquot of the LR reaction was used to transform One Shot TOP10 chemically competent E. coli cells following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA).

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: Sequencing using amplification primers resulted in the characterization of a 10,075 nt sequence. .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen).

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing. .. Alignment and analysis of obtained sequences were carried out using BioEdit, version 7.2.5 ( ).

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: The amplified product was purified using a QIAquick PCR purification kit (Qiagen) and ligated into pBAD TOPO TA (Invitrogen) vector and sequence confirmed. .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression.

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: Transposon insertion, an alternative to subcloning or primer walking when sequencing a large region of DNA, was performed essentially as described elsewhere ( ). .. Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD).

    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. ..

    Cell Culture:

    Article Title: Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
    Article Snippet: After purification of the plasmids from the transformed Top10 competent cells (Invitrogen) the cDNA inserts were transferred to the pAd (adenovirus)/CMV/V5-DEST vector (Invitrogen) by means of the Gateway® system using LR Clonase™ (Invitrogen). .. Then 293A cells were cultured for 1–2 weeks in RPMI 1640 medium containing 10% foetal calf serum, with replacement of the medium every 2 days.

    Generated:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: .. The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells. .. Transformants were selected on low-salt Luria-Bertani plates containing 25 μg/mL zeocin (Duchefa Biochemie) and analyzed by direct PCR amplification using specific primers of the α-factor signal peptide and the 3′-Alcohol Oxidase terminator sequence according to the manufacturer’s instruction.

    Article Title: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA). .. The clone libraries were developed from PCR products generated using fecal and water DNA extracts.

    Article Title: The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem ▿
    Article Snippet: The PmeI-PstI fragments from pRB8531, pRB8532, pRB8533, and pRB8534 were then cloned into SacI-PstI-digested pKO5 and generated pKO8531, pKO8532, pKO8533, and pKO8534. .. Competent cells of Escherichia coli strain RR1 harboring bacterial accessory chromosome 8502 (BAC8502) ( ) were electroporated with pKO8535 and incubated at 43°C on Lennox LB (Invitrogen, Carlsbad, CA) plates containing zeocin (25 μg/ml) and chloramphenicol (20 μg/ml).

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: Fragments C2, C9 and C12 were synthesized using a two-step protocol: cDNA was generated using a mixture of random hexaprimers (RT Taqman Applied Biosystems) followed by PCR amplification using Taq Polymerase (Invitrogen). .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen).

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: Δ hylB mutant was generated by precise in-frame allelic replacement of hylB with chloramphenicol acetyltransferase ( cat ) in the WT GBS strain A909, as previously described ( ). .. This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen).

    Inhibition:

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: To test for inhibition, the snout, dorsal, and ventral skin DNA extracts of four randomly selected PCR- and culture-negative snakes were spiked with 5 pg O. ophiodiicola gDNA. .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions.

    DNA Sequencing:

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: These primer sites were subsequently used for DNA sequencing reactions, making it possible to simultaneously sequence large regions of DNA. .. Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD).

    Polymerase Chain Reaction:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells. .. Transformants were selected on low-salt Luria-Bertani plates containing 25 μg/mL zeocin (Duchefa Biochemie) and analyzed by direct PCR amplification using specific primers of the α-factor signal peptide and the 3′-Alcohol Oxidase terminator sequence according to the manufacturer’s instruction.

    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: Purified PCR products of F1 and F2 were digested with Hin dIII and Kpn I or Kpn I and Eco RI, respectively, for 1 h at 37°C. pcDNA3.1(+) vector was digested with Eco RI and Hin dIII under the same conditions. .. The ligation mixture was then transformed to competent cells (Invitrogen) and screened by ampicillin.

    Article Title: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: .. Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA). .. The clone libraries were developed from PCR products generated using fecal and water DNA extracts.

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: To verify cloning effectiveness, isolated plasmids from 5-ml bacterial cultures were used as template in PCR reactions using both external (cloning) and internal primers, and by digestion with the restriction enzyme Eco RI (Boehringer, Petersburg, VA) and conventional agarose gels. .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used.

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Approximately 40 clones per serum were generated and sequenced using the T7 promoter primer ( 5′-CCCTATAGTGAGTCGTATTA-3′ ).

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing. .. Alignment and analysis of obtained sequences were carried out using BioEdit, version 7.2.5 ( ).

    Article Title: Development of novel O-polysaccharide based glycoconjugates for immunization against glanders
    Article Snippet: PCR and restriction digested products were purified using a QIAquick Gel Extraction Kit (Qiagen). .. Chemically competent E. coli TOP10 cells were transformed as per the manufacturer's instructions (Invitrogen).

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: The amplified product was purified using a QIAquick PCR purification kit (Qiagen) and ligated into pBAD TOPO TA (Invitrogen) vector and sequence confirmed. .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression.

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions. .. Ethics statement All carcass collection, euthanasia, and biopsy sampling procedures were performed under the authority of other agencies in accordance with applicable state laws; specimens were submitted for routine diagnostic analyses that did not require further review by NWHC institutional committees.

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: The fusion product resulting from this PCR contained an in-frame substitution of hylB with cat and was subcloned into the Gateway entry vector pCR8/GW/TOPO. .. This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen).

    Recombinant:

    Article Title: The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem ▿
    Article Snippet: Paragraph title: Construction of recombinant viruses. ... Competent cells of Escherichia coli strain RR1 harboring bacterial accessory chromosome 8502 (BAC8502) ( ) were electroporated with pKO8535 and incubated at 43°C on Lennox LB (Invitrogen, Carlsbad, CA) plates containing zeocin (25 μg/ml) and chloramphenicol (20 μg/ml).

    Article Title: Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion
    Article Snippet: An aliquot of the LR reaction was used to transform One Shot TOP10 chemically competent E. coli cells following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA). .. Recombinant clones were selected on LB agar plates supplemented with 100 μg/mL of ampicillin.

    Article Title: Development of novel O-polysaccharide based glycoconjugates for immunization against glanders
    Article Snippet: Paragraph title: Recombinant DNA techniques ... Chemically competent E. coli TOP10 cells were transformed as per the manufacturer's instructions (Invitrogen).

    Fluorescence:

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used. .. The relative plasmid DNA concentration was determined by fluorescence using the PicoGreen dsDNA quantitation reagent (Molecular Probes, Eugene, OR).

    Mutagenesis:

    Article Title: The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem ▿
    Article Snippet: Mutations were generated with a QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's manual. .. Competent cells of Escherichia coli strain RR1 harboring bacterial accessory chromosome 8502 (BAC8502) ( ) were electroporated with pKO8535 and incubated at 43°C on Lennox LB (Invitrogen, Carlsbad, CA) plates containing zeocin (25 μg/ml) and chloramphenicol (20 μg/ml).

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: Paragraph title: Construction of the Δ hylB mutant ... This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen).

    Isolation:

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: To verify cloning effectiveness, isolated plasmids from 5-ml bacterial cultures were used as template in PCR reactions using both external (cloning) and internal primers, and by digestion with the restriction enzyme Eco RI (Boehringer, Petersburg, VA) and conventional agarose gels. .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used.

    Article Title: Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion
    Article Snippet: An aliquot of the LR reaction was used to transform One Shot TOP10 chemically competent E. coli cells following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA). .. Plasmid DNA was isolated from several colonies using QIAGEN Plasmid Mini Kit (QIAGEN, Valencia, CA) and digested with EcoR I and Kpn I (Promega, Madison, WI) to confirm the presence of the insert in pVP16.

    Article Title: Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation
    Article Snippet: .. p21 sense plasmid DNA Plasmid DNA was isolated from competent E-coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen, Carlsbad CA) or the vector containing the full-length p21WAF1/CIP1 gene in the sense direction described by us [ ]. .. Mixed Lymphocyte Reaction (MLR) MLR was performed with splenocytes from isografts, untreated and CsA treated transplant recipients (LEW) as responders and irradiated splenocytes from donor strain (WF) as stimulators.

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: Isolated DNA was amplified by PCR using the GeneAmp® RNA PCR Core Kit (Applied Biosystems, USA). .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing.

    Subcloning:

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: Transposon insertion, an alternative to subcloning or primer walking when sequencing a large region of DNA, was performed essentially as described elsewhere ( ). .. Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD).

    Purification:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: Paragraph title: FpPG Expression and Purification ... The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells.

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used. .. HPV plasmids were purified using the QIAGEN plasmid Maxi purification kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions.

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Approximately 40 clones per serum were generated and sequenced using the T7 promoter primer ( 5′-CCCTATAGTGAGTCGTATTA-3′ ).

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing. .. Alignment and analysis of obtained sequences were carried out using BioEdit, version 7.2.5 ( ).

    Article Title: Development of novel O-polysaccharide based glycoconjugates for immunization against glanders
    Article Snippet: Genomic DNA was purified using a Wizard Genomic DNA Purification kit (Promega). .. Chemically competent E. coli TOP10 cells were transformed as per the manufacturer's instructions (Invitrogen).

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: The amplified product was purified using a QIAquick PCR purification kit (Qiagen) and ligated into pBAD TOPO TA (Invitrogen) vector and sequence confirmed. .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression.

    Article Title: Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
    Article Snippet: .. After purification of the plasmids from the transformed Top10 competent cells (Invitrogen) the cDNA inserts were transferred to the pAd (adenovirus)/CMV/V5-DEST vector (Invitrogen) by means of the Gateway® system using LR Clonase™ (Invitrogen). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Approximately 40 clones per serum were generated and sequenced using the T7 promoter primer ( 5′-CCCTATAGTGAGTCGTATTA-3′ ).

    Staining:

    Article Title: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: PCR products were visualized in 1.5% agarose gels using GelStar nucleic acid gel stain (Lonza, Rockland, ME). .. Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA).

    IA:

    Article Title: Development of novel O-polysaccharide based glycoconjugates for immunization against glanders
    Article Snippet: Chemically competent E. coli TOP10 cells were transformed as per the manufacturer's instructions (Invitrogen). .. Oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, IA).

    Plasmid Preparation:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells. .. Plasmid DNA was extracted from the cells using a plasmid mini prep kit (Macherey & Nagel) and analyzed by digestion with Eco RI and Xba I restriction enzymes, followed by 1% agarose gel analysis.

    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: Paragraph title: Two Fragments of F1 and F2 and Linearized Vector Were Assembled Sequentially by Ligase to Generate Flag-PTH1R Construct ... The ligation mixture was then transformed to competent cells (Invitrogen) and screened by ampicillin.

    Article Title: Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay
    Article Snippet: .. Domain Bacteria and class Epsilonproteobacteria PCR products were cloned into pCR4 TOPO vector and transformed into TOPO10 chemically competent E. coli cells as described by the manufacturer (Invitrogen, Carlsbad, CA). .. The clone libraries were developed from PCR products generated using fecal and water DNA extracts.

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: Amplified fragments from HPV-16 were cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and the amplified fragments from HPV-18, HPV-45, and HPV-51 were cloned into the pGEM-T easy vector (Promega, Madison, WI) according to the specifications of the manufacturers. .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used.

    Article Title: Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion
    Article Snippet: Cloning and expression of MBP-linker-MPR-TM and MBP-AAA-MPR-TM The coding region of gp41 MPR-TM flanked by a tobacco etch virus (TEV) cleavage site was subcloned from pMistic-MPR-TM [ ] into the pCR8/GW/Topo vector (Invitrogen, Carlsbad, CA), sequence-verified and shuttled into the pVP16 destination vector [ ] by Gateway recombination cloning (Invitrogen, Carlsbad, CA). .. An aliquot of the LR reaction was used to transform One Shot TOP10 chemically competent E. coli cells following the protocol provided by the manufacturer (Invitrogen, Carlsbad, CA).

    Article Title: Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation
    Article Snippet: .. p21 sense plasmid DNA Plasmid DNA was isolated from competent E-coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen, Carlsbad CA) or the vector containing the full-length p21WAF1/CIP1 gene in the sense direction described by us [ ]. .. Mixed Lymphocyte Reaction (MLR) MLR was performed with splenocytes from isografts, untreated and CsA treated transplant recipients (LEW) as responders and irradiated splenocytes from donor strain (WF) as stimulators.

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: .. For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Approximately 40 clones per serum were generated and sequenced using the T7 promoter primer ( 5′-CCCTATAGTGAGTCGTATTA-3′ ).

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing. .. Alignment and analysis of obtained sequences were carried out using BioEdit, version 7.2.5 ( ).

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol
    Article Snippet: The amplified product was purified using a QIAquick PCR purification kit (Qiagen) and ligated into pBAD TOPO TA (Invitrogen) vector and sequence confirmed. .. This construct was transformed into TOP10 competent cells (Invitrogen) for expression.

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD). .. Plasmid DNA was extracted from single “transconjugate” colonies and sequenced using the unique primer sites present on the transposon.

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions. .. Ethics statement All carcass collection, euthanasia, and biopsy sampling procedures were performed under the authority of other agencies in accordance with applicable state laws; specimens were submitted for routine diagnostic analyses that did not require further review by NWHC institutional committees.

    Article Title: Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
    Article Snippet: .. After purification of the plasmids from the transformed Top10 competent cells (Invitrogen) the cDNA inserts were transferred to the pAd (adenovirus)/CMV/V5-DEST vector (Invitrogen) by means of the Gateway® system using LR Clonase™ (Invitrogen). ..

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: .. This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen). .. Plasmid DNA was extracted, and the fusion PCR amplicon was transferred into the temperature-sensitive knockout vector pKODestErm ( ) via an attL–attR (LR) recombination reaction to create the knockout plasmid pKO hylB .

    Software:

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: The sensitivity of each PCR was compared to that of the culture-based analyses with McNemar’s Test in SigmaPlot 11.2 (Systat Software, Inc., San Jose, CA). .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions.

    Selection:

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD). .. Double antibiotic selection was used to isolate transformants having the genomic insert and the incorporated transposon.

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen). .. Transformants were identified at 30°C by Erm selection and shifted to 37°C (a non-permissive temperature for plasmid replication).

    Agarose Gel Electrophoresis:

    Article Title: Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering 1 [W]
    Article Snippet: The construct, generated in frame with the signal sequence for secretion of the yeast ( Saccharomyces cerevisiae ) α factor, was amplified by transforming Escherichia coli DH5α [genotype: F- φ80 lac ZΔM15 Δ( lac ZYA- arg F)U169 deo R rec A1 end A1 hsd R17(rk−, mk+) pho A sup E44 thi -1 gyr A96 rel A1 λ-, provided by Invitrogen] competent cells. .. Plasmid DNA was extracted from the cells using a plasmid mini prep kit (Macherey & Nagel) and analyzed by digestion with Eco RI and Xba I restriction enzymes, followed by 1% agarose gel analysis.

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: The specificity of the amplification was evaluated by agarose-gel electrophoresis and by sequence analysis. .. All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used.

    In Vitro:

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: .. Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD). .. Double antibiotic selection was used to isolate transformants having the genomic insert and the incorporated transposon.

    Chromosome Walking:

    Article Title: Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
    Article Snippet: Transposon insertion, an alternative to subcloning or primer walking when sequencing a large region of DNA, was performed essentially as described elsewhere ( ). .. Briefly, transposons were randomly integrated into the target DNA in an in vitro transposition reaction; the DNA was isopropanol-precipitated and electroporated into competent cells (ElectroMax DH10B, Life Technologies, Rockville, MD).

    Quantitation Assay:

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used. .. The relative plasmid DNA concentration was determined by fluorescence using the PicoGreen dsDNA quantitation reagent (Molecular Probes, Eugene, OR).

    Knock-Out:

    Article Title: Group B Streptococcus evades host immunity by degrading hyaluronan
    Article Snippet: This vector was then used to transform chemically competent Mach 1 E. coli cells (Invitrogen). .. Plasmid DNA was extracted, and the fusion PCR amplicon was transferred into the temperature-sensitive knockout vector pKODestErm ( ) via an attL–attR (LR) recombination reaction to create the knockout plasmid pKO hylB .

    Produced:

    Article Title: Dengue 1 Diversity and Microevolution, French Polynesia 2001-2006: Connection with Epidemiology and Clinics
    Article Snippet: For the analysis of intra-host genetic diversity, the Qiagen OneStep RT-PCR kit was used together with primers Q1F-Q1R ( ) to produce a 758 nt fragment within the E-gene, which was subsequently purified using the QIAquick PCR Purification Kit, ligated into the cloning vector pCR 2.1 and transformed into TOP10 competent cells, according to the manufacturer's protocol (TA Cloning, Invitrogen). .. Serial dilutions were produced and the last dilution providing a clear positive signal was used as a control.

    Article Title: TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease
    Article Snippet: Amplified DNA from 5 randomly selected PCR- and culture-positive samples and all culture-negative samples that produced positive PCR results were sequenced to confirm that the amplicons matched the targeted region. .. Briefly, amplicons were cloned using the Invitrogen™ TOPO® TA Cloning® Kit with the pCR™ 2.1-TOPO® vector and TOP10 competent cells (Life Technologies, Carlsbad, CA), the clones screened by PCR with vector-specific primers, and the resulting PCR products sequenced according to the manufacturer’s instructions.

    Concentration Assay:

    Article Title: Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types
    Article Snippet: All HPV clones were transformed into One Shot Top-10 chemically competent cells (Invitrogen) and stored as glycerol stocks at −20°C until used. .. The relative plasmid DNA concentration was determined by fluorescence using the PicoGreen dsDNA quantitation reagent (Molecular Probes, Eugene, OR).

    DNA Purification:

    Article Title: Prevalence and molecular characterisation of Acanthocephala in pinnipedia of the North and Baltic Seas
    Article Snippet: .. If sequencing of PCR products was not successful, PCR products were ligated into 4-TOPO™ vector and inserted in chemically competent TOP10 E. coli cells (OneShot™ TOP10) using the TOPO™ TA Cloning™ Kit (Life Technologies Inc., USA) and purified plasmids (NucleoSpin™ Plasmid DNA Purification Kit, Macherey-Nagel, Germany) were used for Sanger sequencing. .. Alignment and analysis of obtained sequences were carried out using BioEdit, version 7.2.5 ( ).

    Article Title: Development of novel O-polysaccharide based glycoconjugates for immunization against glanders
    Article Snippet: Genomic DNA was purified using a Wizard Genomic DNA Purification kit (Promega). .. Chemically competent E. coli TOP10 cells were transformed as per the manufacturer's instructions (Invitrogen).

    Gel Extraction:

    Article Title: Development of novel O-polysaccharide based glycoconjugates for immunization against glanders
    Article Snippet: PCR and restriction digested products were purified using a QIAquick Gel Extraction Kit (Qiagen). .. Chemically competent E. coli TOP10 cells were transformed as per the manufacturer's instructions (Invitrogen).

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