ecori restriction enzyme  (Thermo Fisher)


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    Name:
    EcoRI 10 U µL
    Description:
    5 G ↓A A T T C 3 3 C T T A A ↑G 5 Thermo Scientific EcoRI restriction enzyme recognizes G AATTC sites and cuts best at 37°C in its own unique buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Note Also available as a FastDigest enzyme for rapid DNA digestion Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that sent in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    Catalog Number:
    er0271
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    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.
    5 G ↓A A T T C 3 3 C T T A A ↑G 5 Thermo Scientific EcoRI restriction enzyme recognizes G AATTC sites and cuts best at 37°C in its own unique buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Note Also available as a FastDigest enzyme for rapid DNA digestion Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that sent in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    https://www.bioz.com/result/ecori restriction enzyme/product/Thermo Fisher
    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    ecori restriction enzyme - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases"

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

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.08.015

    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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

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

    Related Articles

    Construct:

    Article Title: Plasmodium vivax Ookinete Surface Protein Pvs25 Linked to Cholera Toxin B Subunit Induces Potent Transmission-Blocking Immunity by Intranasal as Well as Subcutaneous Immunization ▿
    Article Snippet: .. The fragment was subcloned into the SnaBI and EcoRI sites of the P. pastoris expression vector pPIC9K (Life Technologies, Carlsbad, CA) to construct the plasmid pPvs25H, which was designed to express an α-factor signal-Pvs25-hexahistidine fusion protein (see Fig. ). .. P. pastoris recombination was performed according to the manufacturer's instructions (Life Technologies).

    Purification:

    Article Title: Genome Analysis of Lactobacillus plantarum LL441 and Genetic Characterisation of the Locus for the Lantibiotic Plantaricin C
    Article Snippet: .. Briefly, total genomic DNA was extracted and purified from L. plantarum LL441, and then digested with PstI, EcoRI, or HindIII (Fermentas GmbH, Sankt Leon-Rot, Germany). .. After transferring to a Hybond nylon membrane (Amersham; GE Healthcare, Pittsburgh, PA., United States), the DNA was hybridized with a probe based on an internal segment of the coding sequence of the lanM gene amplified by PCR.

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. Phage DNA was digested using EcoRI (Invitrogen), separated on 0.8% (wt/vol) agarose gels, purified using the GeneClean II kit (Qbiogene, Irvine, CA), ligated into pUC19 or pGEM-7Z and transformed into DH5α (Invitrogen). .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis.

    Generated:

    Article Title: Human Mre11/Human Rad50/Nbs1 and DNA Ligase III?/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway *
    Article Snippet: .. Linear DNA molecules were generated by digestion of pGADT7 (Invitrogen) with BamHI and EcoRI for the four nucleotide 5′ overhangs and of pcDNA4HisMax C (Invitrogen) with KpnI and PstI for the four nucleotide 3′ overhangs. .. Intramolecular joining of the incompatible ends was carried out in 25 m m MOPS (pH 7.0), 60 m m KCl, 0.2% Tween 20, 2 m m DTT, 4 m m MgCl2 , 2 m m MnCl2 , 0.5 m m ATP, 0.8 pmol of plasmid DNA, 10% polyethylene glycol, 0.01 pmol of DNA ligase III/XRCC1 or DNA ligase IV/XRCC4, and 0.06 pmol of hMre11 or MRN as indicated, in a volume of 10 μl.

    Expressing:

    Article Title: Plasmodium vivax Ookinete Surface Protein Pvs25 Linked to Cholera Toxin B Subunit Induces Potent Transmission-Blocking Immunity by Intranasal as Well as Subcutaneous Immunization ▿
    Article Snippet: .. The fragment was subcloned into the SnaBI and EcoRI sites of the P. pastoris expression vector pPIC9K (Life Technologies, Carlsbad, CA) to construct the plasmid pPvs25H, which was designed to express an α-factor signal-Pvs25-hexahistidine fusion protein (see Fig. ). .. P. pastoris recombination was performed according to the manufacturer's instructions (Life Technologies).

    Polymerase Chain Reaction:

    Article Title: Allelic Variations of Plasmodium vivax Apical Membrane Antigen-1 (Pv AMA-1) in Malarious Areas of Southeastern Iran Using PCR-RFLP Technique
    Article Snippet: .. In order to determine the presence of different alleles of Pv AMA-1 gene in the region, PCR–RFLP technique was done to digest the gene using three restriction enzymes EcoR-1, Pvu-II and Hind3 (Thermo cat No #ER0271, #ER0631 and ferments cat No #ER0501 respectively) according to the manufacturer’s recommendations. .. The products were visualized under UV illumination after electrophoresis on 3% agarose gel containing ethidium bromide.

    Article Title: Allelic Variations of Plasmodium vivax Apical Membrane Antigen-1 (Pv AMA-1) in Malarious Areas of Southeastern Iran Using PCR-RFLP Technique
    Article Snippet: .. RFLP In order to determine the presence of different alleles of Pv AMA-1 gene in the region, PCR–RFLP technique was done to digest the gene using three restriction enzymes EcoR-1, Pvu-II and Hind3 (Thermo cat No #ER0271, #ER0631 and ferments cat No #ER0501 respectively) according to the manufacturer’s recommendations. .. The products were visualized under UV illumination after electrophoresis on 3% agarose gel containing ethidium bromide.

    Transformation Assay:

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. Phage DNA was digested using EcoRI (Invitrogen), separated on 0.8% (wt/vol) agarose gels, purified using the GeneClean II kit (Qbiogene, Irvine, CA), ligated into pUC19 or pGEM-7Z and transformed into DH5α (Invitrogen). .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis.

    Plasmid Preparation:

    Article Title: Plasmodium vivax Ookinete Surface Protein Pvs25 Linked to Cholera Toxin B Subunit Induces Potent Transmission-Blocking Immunity by Intranasal as Well as Subcutaneous Immunization ▿
    Article Snippet: .. The fragment was subcloned into the SnaBI and EcoRI sites of the P. pastoris expression vector pPIC9K (Life Technologies, Carlsbad, CA) to construct the plasmid pPvs25H, which was designed to express an α-factor signal-Pvs25-hexahistidine fusion protein (see Fig. ). .. P. pastoris recombination was performed according to the manufacturer's instructions (Life Technologies).

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli
    Article Snippet: .. The Modular Sender Vector and each HSL synthase oligo were cut with EcoRI and XbaI (Thermo Fisher Scientific) and ligated using T4 ligase (New England Biolabs). ..

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  • 99
    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 19 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 19 article reviews
    Price from $9.99 to $1999.99
    ecori restriction enzyme - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    90
    Thermo Fisher restriction enzymes ecori
    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.
    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, 2020-08
    90/100 stars
      Buy from Supplier

    Image Search Results


    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