ecorv enzyme  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ecorv enzyme
    Ecorv Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecorv enzyme/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ecorv enzyme - by Bioz Stars, 2020-07
    93/100 stars

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    Restriction Assay:

    Article Title: CXCL4 assembles DNA into liquid crystalline complexes to amplify TLR9-mediated interferon-α production in systemic sclerosis
    Article Snippet: .. Restriction protection assay Restriction assay solution (DPBS with 0.5 mM MgCl2 , 0.9 mM CaCl2 , and 0.1 mg/ml BSA) containing 150 ng (1.97 nM) of circular pDB29 DNA was incubated for 30 min at 37 °C with various concentrations of CXCL4 or control molecules in the presence/absence of 0.55 U of EcoRV enzyme (ThermoFisher Scientific). .. DNA products were extracted with phenol/CHCl3 , ethanol precipitated, and electrophoresed on a standard 1% agarose gel in 1× TAE with ethidium bromide.

    Incubation:

    Article Title: CXCL4 assembles DNA into liquid crystalline complexes to amplify TLR9-mediated interferon-α production in systemic sclerosis
    Article Snippet: .. Restriction protection assay Restriction assay solution (DPBS with 0.5 mM MgCl2 , 0.9 mM CaCl2 , and 0.1 mg/ml BSA) containing 150 ng (1.97 nM) of circular pDB29 DNA was incubated for 30 min at 37 °C with various concentrations of CXCL4 or control molecules in the presence/absence of 0.55 U of EcoRV enzyme (ThermoFisher Scientific). .. DNA products were extracted with phenol/CHCl3 , ethanol precipitated, and electrophoresed on a standard 1% agarose gel in 1× TAE with ethidium bromide.

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    Thermo Fisher ecori restriction enzyme
    Integration and HDR Analysis of CRISPR/Cas9-Edited EBS hKc After co-transfection of the respective Cas9/sgRNA combinations (spCas9-sgRNA1, spCas9-sgRNA2, or both Cas9n-sgRNA1 and Cas9n-sgRNA2) and the MC donor plasmid into EBS hKc, we performed two rounds of blasticidin selection. The resulting blasticidin-resistant cells were then analyzed for site-specific integration of the donor template at the genomic level. (A) Integration-specific <t>PCR</t> using a forward primer binding the restriction sites <t>EcoRI</t> and NheI (white box) provided by the MC and a reverse primer binding downstream to the 3′ UTR of the KRT14 gene resulted in a PCR product of 893 bp. (B) EcoRI digestion to estimate the relative recombination efficiency in the treated cell pool. We performed a PCR using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 3′ UTR. The PCR resulted in a specific product of 1,189 bp. EcoRI digestion generated the expected fragments of 874 and 315 bp, only visible in the Cas9n 1- and 2-treated EBS hKc. (C) Schematic depiction of the primer binding sites used for amplification of the integration-specific PCR fragments. Gray arrows indicate a forward primer binding to the restriction sites EcoRI and NheI integrated via HDR. Black arrows indicate forward and reverse primers binding to endogenous KRT14 . Black star: missense mutation (c.1231G > A) in exon 6; gray stars: silent mutations.
    Ecori Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecori restriction enzyme/product/Thermo Fisher
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    ecori restriction enzyme - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    89
    Thermo Fisher ecor v enzyme
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Ecor V Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecor v enzyme/product/Thermo Fisher
    Average 89 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    ecor v enzyme - by Bioz Stars, 2020-07
    89/100 stars
      Buy from Supplier

    90
    Thermo Fisher restriction enzymes ecori
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Restriction Enzymes Ecori, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzymes ecori/product/Thermo Fisher
    Average 90 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    restriction enzymes ecori - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

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    Integration and HDR Analysis of CRISPR/Cas9-Edited EBS hKc After co-transfection of the respective Cas9/sgRNA combinations (spCas9-sgRNA1, spCas9-sgRNA2, or both Cas9n-sgRNA1 and Cas9n-sgRNA2) and the MC donor plasmid into EBS hKc, we performed two rounds of blasticidin selection. The resulting blasticidin-resistant cells were then analyzed for site-specific integration of the donor template at the genomic level. (A) Integration-specific PCR using a forward primer binding the restriction sites EcoRI and NheI (white box) provided by the MC and a reverse primer binding downstream to the 3′ UTR of the KRT14 gene resulted in a PCR product of 893 bp. (B) EcoRI digestion to estimate the relative recombination efficiency in the treated cell pool. We performed a PCR using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 3′ UTR. The PCR resulted in a specific product of 1,189 bp. EcoRI digestion generated the expected fragments of 874 and 315 bp, only visible in the Cas9n 1- and 2-treated EBS hKc. (C) Schematic depiction of the primer binding sites used for amplification of the integration-specific PCR fragments. Gray arrows indicate a forward primer binding to the restriction sites EcoRI and NheI integrated via HDR. Black arrows indicate forward and reverse primers binding to endogenous KRT14 . Black star: missense mutation (c.1231G > A) in exon 6; gray stars: silent mutations.

    Journal: Molecular Therapy

    Article Title: Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

    doi: 10.1016/j.ymthe.2017.08.015

    Figure Lengend Snippet: Integration and HDR Analysis of CRISPR/Cas9-Edited EBS hKc After co-transfection of the respective Cas9/sgRNA combinations (spCas9-sgRNA1, spCas9-sgRNA2, or both Cas9n-sgRNA1 and Cas9n-sgRNA2) and the MC donor plasmid into EBS hKc, we performed two rounds of blasticidin selection. The resulting blasticidin-resistant cells were then analyzed for site-specific integration of the donor template at the genomic level. (A) Integration-specific PCR using a forward primer binding the restriction sites EcoRI and NheI (white box) provided by the MC and a reverse primer binding downstream to the 3′ UTR of the KRT14 gene resulted in a PCR product of 893 bp. (B) EcoRI digestion to estimate the relative recombination efficiency in the treated cell pool. We performed a PCR using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 3′ UTR. The PCR resulted in a specific product of 1,189 bp. EcoRI digestion generated the expected fragments of 874 and 315 bp, only visible in the Cas9n 1- and 2-treated EBS hKc. (C) Schematic depiction of the primer binding sites used for amplification of the integration-specific PCR fragments. Gray arrows indicate a forward primer binding to the restriction sites EcoRI and NheI integrated via HDR. Black arrows indicate forward and reverse primers binding to endogenous KRT14 . Black star: missense mutation (c.1231G > A) in exon 6; gray stars: silent mutations.

    Article Snippet: For estimation of the HDR efficiency, we PCR-amplified the KRT14 target region using a forward primer binding to KRT14 exon 6 (5′-CAGGAGATGATTGGCAGCGTGG-3′) and again a reverse primer binding to the endogenous 3′ downstream sequence (5′-GATGCTTCTCCCACTTTCTCCCC-3′) of the KRT14 gene, resulting in a PCR product of 1,189 bp, which was subsequently digested with EcoRI restriction enzyme (Thermo Fisher).

    Techniques: CRISPR, Cotransfection, Plasmid Preparation, Selection, Polymerase Chain Reaction, Binding Assay, Sequencing, Generated, Amplification, Mutagenesis

    Analysis of HDR Efficiency (A) PCR analysis of genomic DNA from CRISPR/Cas9-treated EBS hKc using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 . To minimize the size of the PCR product and to facilitate subcloning, we performed a nested PCR using a forward primer binding to exon 6 and a reverse primer binding to intron 7, resulting in a specific product of 507 bp. An EcoRI restriction digest confirmed the presence of recombined KRT14 alleles in treated EBS cells. Upon bacterial transformation, single colonies were picked for colony PCR and amplified product digested with EcoRI to identify recombined alleles. (B) Sequence analysis of one representative single clone, containing a modified wild-type allele (green arrow: wild-type sequence at mutation site [position 178] within exon 6), is shown. The allele additionally contains the introduced restriction sites NheI/EcoRI and the silent mutations (blue stars). Sequence alignment was performed with the software “Multiple sequence alignment with hierarchical clustering.” 21

    Journal: Molecular Therapy

    Article Title: Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

    doi: 10.1016/j.ymthe.2017.08.015

    Figure Lengend Snippet: Analysis of HDR Efficiency (A) PCR analysis of genomic DNA from CRISPR/Cas9-treated EBS hKc using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 . To minimize the size of the PCR product and to facilitate subcloning, we performed a nested PCR using a forward primer binding to exon 6 and a reverse primer binding to intron 7, resulting in a specific product of 507 bp. An EcoRI restriction digest confirmed the presence of recombined KRT14 alleles in treated EBS cells. Upon bacterial transformation, single colonies were picked for colony PCR and amplified product digested with EcoRI to identify recombined alleles. (B) Sequence analysis of one representative single clone, containing a modified wild-type allele (green arrow: wild-type sequence at mutation site [position 178] within exon 6), is shown. The allele additionally contains the introduced restriction sites NheI/EcoRI and the silent mutations (blue stars). Sequence alignment was performed with the software “Multiple sequence alignment with hierarchical clustering.” 21

    Article Snippet: For estimation of the HDR efficiency, we PCR-amplified the KRT14 target region using a forward primer binding to KRT14 exon 6 (5′-CAGGAGATGATTGGCAGCGTGG-3′) and again a reverse primer binding to the endogenous 3′ downstream sequence (5′-GATGCTTCTCCCACTTTCTCCCC-3′) of the KRT14 gene, resulting in a PCR product of 1,189 bp, which was subsequently digested with EcoRI restriction enzyme (Thermo Fisher).

    Techniques: Polymerase Chain Reaction, CRISPR, Binding Assay, Sequencing, Subcloning, Nested PCR, Electroporation Bacterial Transformation, Amplification, Modification, Mutagenesis, Software

    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Journal: PLoS ONE

    Article Title: Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments

    doi: 10.1371/journal.pone.0116917

    Figure Lengend Snippet: Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Article Snippet: Since the cloned HA-segments of H9N2/SA showed instability in the genetic backbone of pCR2.1 sequencing vector, the full-length RT-PCR products of HA segments amplified with primers containing EcoRV and Bsa I sites were digested with EcoR V enzyme (Thermo Scientific, USA) for 1 h at 37°C to produce blunt-ends to allow the ligation into the EcoRV -linearized pSMART-LC-Kan (Lucigen, USA).

    Techniques: Plasmid Preparation, Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction