target dna  (New England Biolabs)


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    Structured Review

    New England Biolabs target dna
    All six sgRNA produce <t>Cas9-driven</t> cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). <t>DNA</t> bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Target Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9"

    Article Title: Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006769

    All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Figure Legend Snippet: All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Incubation

    2) Product Images from "RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells"

    Article Title: RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

    Journal: bioRxiv

    doi: 10.1101/848309

    CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.
    Figure Legend Snippet: CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.

    Techniques Used: CRISPR, Binding Assay, Agarose Gel Electrophoresis, Sequencing, Generated

    Functional testing of dCas9 fusions. (A) Numbers of puromycin-resistant colonies in the transposition assay. The dCas9-SB100X fusion protein catalyzes ∼30% as many integration events as unfused SB100X transposase (n=3, biological replicates, * p ≤0.05, *** p ≤0.001, error bars represent SEM). ( B) EMSA with dCas9-N57 fusion proteins. dCas9 serves as negative control, N57 as positive control. Binding can be detected for dCas9-N57, but not for N57-dCas9. ( C) Numbers of 6-TG resistant colonies after Cas9 cleavage assay. No disruption of the HPRT gene, as measured by 6-TG resistance, can be detected without the addition of the HPRT sgRNA. In the presence of a HPRT sgRNA, all Cas9 constructs cause significant disruption of the HPRT gene (n=2, biological replicates, * p ≤0.05, ** p ≤0.01, error bars represent SEM).
    Figure Legend Snippet: Functional testing of dCas9 fusions. (A) Numbers of puromycin-resistant colonies in the transposition assay. The dCas9-SB100X fusion protein catalyzes ∼30% as many integration events as unfused SB100X transposase (n=3, biological replicates, * p ≤0.05, *** p ≤0.001, error bars represent SEM). ( B) EMSA with dCas9-N57 fusion proteins. dCas9 serves as negative control, N57 as positive control. Binding can be detected for dCas9-N57, but not for N57-dCas9. ( C) Numbers of 6-TG resistant colonies after Cas9 cleavage assay. No disruption of the HPRT gene, as measured by 6-TG resistance, can be detected without the addition of the HPRT sgRNA. In the presence of a HPRT sgRNA, all Cas9 constructs cause significant disruption of the HPRT gene (n=2, biological replicates, * p ≤0.05, ** p ≤0.01, error bars represent SEM).

    Techniques Used: Functional Assay, Negative Control, Positive Control, Binding Assay, Cleavage Assay, Construct

    3) Product Images from "RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells"

    Article Title: RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

    Journal: bioRxiv

    doi: 10.1101/848309

    CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.
    Figure Legend Snippet: CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.

    Techniques Used: CRISPR, Binding Assay, Agarose Gel Electrophoresis, Sequencing, Generated

    Functional testing of dCas9 fusions. (A) Numbers of puromycin-resistant colonies in the transposition assay. The dCas9-SB100X fusion protein catalyzes ∼30% as many integration events as unfused SB100X transposase (n=3, biological replicates, * p ≤0.05, *** p ≤0.001, error bars represent SEM). ( B) EMSA with dCas9-N57 fusion proteins. dCas9 serves as negative control, N57 as positive control. Binding can be detected for dCas9-N57, but not for N57-dCas9. ( C) Numbers of 6-TG resistant colonies after Cas9 cleavage assay. No disruption of the HPRT gene, as measured by 6-TG resistance, can be detected without the addition of the HPRT sgRNA. In the presence of a HPRT sgRNA, all Cas9 constructs cause significant disruption of the HPRT gene (n=2, biological replicates, * p ≤0.05, ** p ≤0.01, error bars represent SEM).
    Figure Legend Snippet: Functional testing of dCas9 fusions. (A) Numbers of puromycin-resistant colonies in the transposition assay. The dCas9-SB100X fusion protein catalyzes ∼30% as many integration events as unfused SB100X transposase (n=3, biological replicates, * p ≤0.05, *** p ≤0.001, error bars represent SEM). ( B) EMSA with dCas9-N57 fusion proteins. dCas9 serves as negative control, N57 as positive control. Binding can be detected for dCas9-N57, but not for N57-dCas9. ( C) Numbers of 6-TG resistant colonies after Cas9 cleavage assay. No disruption of the HPRT gene, as measured by 6-TG resistance, can be detected without the addition of the HPRT sgRNA. In the presence of a HPRT sgRNA, all Cas9 constructs cause significant disruption of the HPRT gene (n=2, biological replicates, * p ≤0.05, ** p ≤0.01, error bars represent SEM).

    Techniques Used: Functional Assay, Negative Control, Positive Control, Binding Assay, Cleavage Assay, Construct

    4) Product Images from "Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9"

    Article Title: Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006769

    Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Figure Legend Snippet: Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Techniques Used: Purification, Recombinant, Affinity Chromatography, Staining, Nucleic Acid Electrophoresis, Chromatography, Molecular Weight, Produced, Activity Assay, Polymerase Chain Reaction, Incubation

    All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Figure Legend Snippet: All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Incubation

    5) Product Images from "RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells"

    Article Title: RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

    Journal: bioRxiv

    doi: 10.1101/848309

    CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.
    Figure Legend Snippet: CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.

    Techniques Used: CRISPR, Binding Assay, Agarose Gel Electrophoresis, Sequencing, Generated

    Functional testing of dCas9 fusions. (A) Numbers of puromycin-resistant colonies in the transposition assay. The dCas9-SB100X fusion protein catalyzes ∼30% as many integration events as unfused SB100X transposase (n=3, biological replicates, * p ≤0.05, *** p ≤0.001, error bars represent SEM). ( B) EMSA with dCas9-N57 fusion proteins. dCas9 serves as negative control, N57 as positive control. Binding can be detected for dCas9-N57, but not for N57-dCas9. ( C) Numbers of 6-TG resistant colonies after Cas9 cleavage assay. No disruption of the HPRT gene, as measured by 6-TG resistance, can be detected without the addition of the HPRT sgRNA. In the presence of a HPRT sgRNA, all Cas9 constructs cause significant disruption of the HPRT gene (n=2, biological replicates, * p ≤0.05, ** p ≤0.01, error bars represent SEM).
    Figure Legend Snippet: Functional testing of dCas9 fusions. (A) Numbers of puromycin-resistant colonies in the transposition assay. The dCas9-SB100X fusion protein catalyzes ∼30% as many integration events as unfused SB100X transposase (n=3, biological replicates, * p ≤0.05, *** p ≤0.001, error bars represent SEM). ( B) EMSA with dCas9-N57 fusion proteins. dCas9 serves as negative control, N57 as positive control. Binding can be detected for dCas9-N57, but not for N57-dCas9. ( C) Numbers of 6-TG resistant colonies after Cas9 cleavage assay. No disruption of the HPRT gene, as measured by 6-TG resistance, can be detected without the addition of the HPRT sgRNA. In the presence of a HPRT sgRNA, all Cas9 constructs cause significant disruption of the HPRT gene (n=2, biological replicates, * p ≤0.05, ** p ≤0.01, error bars represent SEM).

    Techniques Used: Functional Assay, Negative Control, Positive Control, Binding Assay, Cleavage Assay, Construct

    6) Product Images from "Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9"

    Article Title: Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006769

    All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Figure Legend Snippet: All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Incubation

    Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Figure Legend Snippet: Purification of Cas9 recombinant protein. (A) Purification of Cas9 by affinity chromatography using a Nickel-charged HiTrap Chelating HP. Gradient of Imidazole is indicated by the green line. (B) Coomassie-stained gel electrophoresis of peaks 1 and 2 after affinity chromatography (26 μl of each fraction) shows that peak 2 corresponds to Cas9. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (C) Purification of Cas9 by cation-exchange chromatography using a MonoS 5/50 GL column. Gradient of NaCl is indicated by the green line. (D) Coomassie-stained gel electrophoresis of fractions correspondent to different peaks after cation-exchange chromatography (26 μl of each fraction, peaks 1–4: lanes 1–4). All peaks showed a band of the correct Cas9 molecular weight. Only the majoritarian peak (#2) was collected. Lane 5 corresponds to purified Cas9 after dialysis. M: SeeBlue Plus2 Pre-Stained Protein Standard (Life Technologies). (E) Purification of recombinant Cas9 produced in our laboratory showed endonuclease activity comparable to a commercial Cas9 (PNABio). 200 ng of PCR product of target gene was incubated with each individual sgRNA (lanes 1–12; 3.2 μM) in the presence of 3.8 μM Cas9 protein either purchased from PNABio (upper gel) or obtained in our laboratory (lower gel). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Techniques Used: Purification, Recombinant, Affinity Chromatography, Staining, Nucleic Acid Electrophoresis, Chromatography, Molecular Weight, Produced, Activity Assay, Polymerase Chain Reaction, Incubation

    7) Product Images from "RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells"

    Article Title: RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

    Journal: bioRxiv

    doi: 10.1101/848309

    CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.
    Figure Legend Snippet: CRISPR/Cas components and their validation for transposon targeting. (A) Schematic exon-intron structure of the HPRT gene and positions of the sgRNA binding sites. ( B) Structure of an Alu element and relative positions of sgRNA binding sites. ( C) Number of 6-TG resistant colonies after treatment with Cas9 and HPRT -directed sgRNAs. Significance is calculated in comparison to the -sgRNA sample (n=2, biological replicates for all samples, * p ≤0.05, error bars represent SEM). ( D) Agarose gel electrophoresis of HEK293T gDNA digested with Cas9 and Alu Y-directed sgRNAs. ( E) Sequence logo generated by aligning sequenced gDNA ends after fragmentation with Cas9 and the Alu Y-directed sgRNA (the sequence represents the top strand targeted by the sgRNA). The position of the sgRNA-binding site and PAM is indicated on the top, the cleavage site is marked by the gray arrow. The sequence upstream of the cleavage site is generated from 12 individual sequences, the sequence downstream is generated from 19 individual sequences. The lower sequence represents the Alu Y consensus sequence.

    Techniques Used: CRISPR, Binding Assay, Agarose Gel Electrophoresis, Sequencing, Generated

    8) Product Images from "Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9"

    Article Title: Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006769

    All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.
    Figure Legend Snippet: All six sgRNA produce Cas9-driven cleavage in vitro . 200 ng of the PCR product of the LuloYLW gene exon 3 were incubated with each individual sgRNA (3.2 μM) in the absence (lanes 1–6) or presence of 3.8 μM Cas9 protein (lanes 7–12). DNA bands size corresponds to the expected size according to each cleavage site (sgRNA1 = 218 bp, 154 bp; sgRNA2 = 230 bp, 142 bp; sgRNA3 = 294 bp, 78 bp; sgRNA4 = 279 bp, 93; sgRNA5 = 158 bp, 214 bp and sgRNA6 = 148 bp, 224 bp). As negative controls, 200 ng of PCR product alone (lane 13) or in combination with Cas9 protein (lane 14) were included. All samples were run on 2.2% agarose gels and visualized under UV light.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Incubation

    Related Articles

    Clone Assay:

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers
    Article Snippet: .. For deletion of the e3 enhancer, tandem U6-promoter-sgRNA and H1-promoter-sgRNA cassettes were cloned into lentiCRISPR_v2 (Addgene #60954) for single vector expression of two sgRNAs as follows: 1) U6-sgRNA and H1-sgRNA products were generated by PCR amplification using the primers listed in , 2) PCR products were then digested with BsmBI to generate compatible sticky ends, 3) finally, three-way ligation of the two PCR products and BsmBI-digested lentiCRISPR_v2 was performed using T7 DNA ligase (NEB #M0318). .. Control ‘empty’ lentiCRISPR_v2 lacking expression of any sgRNAs was generated by BsmBI digestion, followed by blunting of ends (NEB #E1201) and ligation with T4 DNA ligase (NEB #M0202).

    Article Title: Gene essentiality landscape and druggable oncogenic dependencies in herpesviral primary effusion lymphoma
    Article Snippet: .. Cloning of individual sgRNA constructs All sgRNAs were cloned into plentiGuide-Puro using the BsmBI cloning site (New England Biolabs, Ipswich, MA). sgRNA sequences targeting specific genes were designed using publicly available tools , or were derived from the Brunello library. sgRNA sequences and sequences of primers used for cloning are listed in Supplementary Data . .. Lentivirus preparation For library production, 107 HEK-293T cells were seeded per 15 cm dish the day before transfection.

    Amplification:

    Article Title: Programmed Death Receptor 1 (PD1) Knockout and Human Telomerase Reverse Transcriptase (hTERT) Transduction Can Enhance Persistence and Antitumor Efficacy of Cytokine-Induced Killer Cells Against Hepatocellular Carcinoma
    Article Snippet: .. After the i n vitro transcription template of T7-sgRNAs was amplified by PCR, the sgRNAs were transcribed in vitro using a HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, US). .. The in vitro transcription single-guide RNAs (IVT sgRNAs) were purified by using RNA clean & concentratorTM-25 (Zymo Research, US), eluted in RNase-free water, and used immediately after elution or stored at −80°C.

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers
    Article Snippet: .. For deletion of the e3 enhancer, tandem U6-promoter-sgRNA and H1-promoter-sgRNA cassettes were cloned into lentiCRISPR_v2 (Addgene #60954) for single vector expression of two sgRNAs as follows: 1) U6-sgRNA and H1-sgRNA products were generated by PCR amplification using the primers listed in , 2) PCR products were then digested with BsmBI to generate compatible sticky ends, 3) finally, three-way ligation of the two PCR products and BsmBI-digested lentiCRISPR_v2 was performed using T7 DNA ligase (NEB #M0318). .. Control ‘empty’ lentiCRISPR_v2 lacking expression of any sgRNAs was generated by BsmBI digestion, followed by blunting of ends (NEB #E1201) and ligation with T4 DNA ligase (NEB #M0202).

    In Vitro:

    Article Title: Programmed Death Receptor 1 (PD1) Knockout and Human Telomerase Reverse Transcriptase (hTERT) Transduction Can Enhance Persistence and Antitumor Efficacy of Cytokine-Induced Killer Cells Against Hepatocellular Carcinoma
    Article Snippet: .. After the i n vitro transcription template of T7-sgRNAs was amplified by PCR, the sgRNAs were transcribed in vitro using a HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, US). .. The in vitro transcription single-guide RNAs (IVT sgRNAs) were purified by using RNA clean & concentratorTM-25 (Zymo Research, US), eluted in RNase-free water, and used immediately after elution or stored at −80°C.

    Article Title: DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes
    Article Snippet: .. To prepare the RNP complexes, the purified Cas9 protein (0 to 60 μg) was mixed with in vitro -transcribed sgRNA (0 to 60 μg) in NEBuffer 3 and incubated for 15 min at 25°C. .. They were mixed with protoplast suspensions before addition of an equal volume of 40% PEG 4000, then mixed gently and incubated at room temperature in the dark for 15 min. An equal volume of W5 solution was added twice, then mixed and centrifuged at 80 × g for 5 min.

    Article Title: CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii
    Article Snippet: .. For in vitro assays, 500 ng of sgRNA and 600 ng of Cas9 protein were pre-mixed at 37 °C for 5 min. For experiments, 100 ng of PCR amplicons and 3.1 NEB buffer (NEB, USA) were added, and the mixtures were incubated at 37 °C for 2 hours. .. RNaseA (Bioneer, Republic of Korea) was added, and the mixture was incubated at 37 °C for 20 min. 6× STOP solution (0.5 M EDTA, 80% glycerol, and 10% SDS) was added, and the mixture was incubated 37 °C for 20 min.

    Ligation:

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers
    Article Snippet: .. For deletion of the e3 enhancer, tandem U6-promoter-sgRNA and H1-promoter-sgRNA cassettes were cloned into lentiCRISPR_v2 (Addgene #60954) for single vector expression of two sgRNAs as follows: 1) U6-sgRNA and H1-sgRNA products were generated by PCR amplification using the primers listed in , 2) PCR products were then digested with BsmBI to generate compatible sticky ends, 3) finally, three-way ligation of the two PCR products and BsmBI-digested lentiCRISPR_v2 was performed using T7 DNA ligase (NEB #M0318). .. Control ‘empty’ lentiCRISPR_v2 lacking expression of any sgRNAs was generated by BsmBI digestion, followed by blunting of ends (NEB #E1201) and ligation with T4 DNA ligase (NEB #M0202).

    Mutagenesis:

    Article Title: Actomyosin regulation by Eph receptor signaling couples boundary cell formation to border sharpness
    Article Snippet: .. To introduce the K658M mutation in the kinase domain of EphA4a, sgRNA and Cas9 protein were co-injected with a 74 bp donor oligonucleotide (AAGATGCCTGGAAA GCGTGAaATtTGcGTGGCCATAAAAACCCTAAtGGCAGGgTACACCGACAAGCAAAGGCG) containing three silent mutations at the gRNA target site, the K658M mutation and an additional silent mutation that generated an RsaI restriction site. .. Mutations were identified by amplicon restriction using restriction enzymes or T7 endonuclease I (#M0302L NEB) and verified by sequencing.

    Incubation:

    Article Title: DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes
    Article Snippet: .. To prepare the RNP complexes, the purified Cas9 protein (0 to 60 μg) was mixed with in vitro -transcribed sgRNA (0 to 60 μg) in NEBuffer 3 and incubated for 15 min at 25°C. .. They were mixed with protoplast suspensions before addition of an equal volume of 40% PEG 4000, then mixed gently and incubated at room temperature in the dark for 15 min. An equal volume of W5 solution was added twice, then mixed and centrifuged at 80 × g for 5 min.

    Article Title: Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
    Article Snippet: .. Target DNA (200 ng) was mixed to pre-loaded Cas9 protein and reactions were incubated at 37 °C in the presence of 1X Bovine Serum Albumin and 1X NEB3 buffer (New England Biolabs) in a total volume of 20 μl. .. After 1 h and 15 min incubation period, Cas9 protein was inactivated at 65 °C for 10 min. As controls, template DNA were incubated with individual sgRNA in the absence of Cas9 protein.

    Article Title: CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii
    Article Snippet: .. For in vitro assays, 500 ng of sgRNA and 600 ng of Cas9 protein were pre-mixed at 37 °C for 5 min. For experiments, 100 ng of PCR amplicons and 3.1 NEB buffer (NEB, USA) were added, and the mixtures were incubated at 37 °C for 2 hours. .. RNaseA (Bioneer, Republic of Korea) was added, and the mixture was incubated at 37 °C for 20 min. 6× STOP solution (0.5 M EDTA, 80% glycerol, and 10% SDS) was added, and the mixture was incubated 37 °C for 20 min.

    Introduce:

    Article Title: Actomyosin regulation by Eph receptor signaling couples boundary cell formation to border sharpness
    Article Snippet: .. To introduce the K658M mutation in the kinase domain of EphA4a, sgRNA and Cas9 protein were co-injected with a 74 bp donor oligonucleotide (AAGATGCCTGGAAA GCGTGAaATtTGcGTGGCCATAAAAACCCTAAtGGCAGGgTACACCGACAAGCAAAGGCG) containing three silent mutations at the gRNA target site, the K658M mutation and an additional silent mutation that generated an RsaI restriction site. .. Mutations were identified by amplicon restriction using restriction enzymes or T7 endonuclease I (#M0302L NEB) and verified by sequencing.

    Purification:

    Article Title: DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes
    Article Snippet: .. To prepare the RNP complexes, the purified Cas9 protein (0 to 60 μg) was mixed with in vitro -transcribed sgRNA (0 to 60 μg) in NEBuffer 3 and incubated for 15 min at 25°C. .. They were mixed with protoplast suspensions before addition of an equal volume of 40% PEG 4000, then mixed gently and incubated at room temperature in the dark for 15 min. An equal volume of W5 solution was added twice, then mixed and centrifuged at 80 × g for 5 min.

    Plasmid Preparation:

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers
    Article Snippet: .. For deletion of the e3 enhancer, tandem U6-promoter-sgRNA and H1-promoter-sgRNA cassettes were cloned into lentiCRISPR_v2 (Addgene #60954) for single vector expression of two sgRNAs as follows: 1) U6-sgRNA and H1-sgRNA products were generated by PCR amplification using the primers listed in , 2) PCR products were then digested with BsmBI to generate compatible sticky ends, 3) finally, three-way ligation of the two PCR products and BsmBI-digested lentiCRISPR_v2 was performed using T7 DNA ligase (NEB #M0318). .. Control ‘empty’ lentiCRISPR_v2 lacking expression of any sgRNAs was generated by BsmBI digestion, followed by blunting of ends (NEB #E1201) and ligation with T4 DNA ligase (NEB #M0202).

    Generated:

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers
    Article Snippet: .. For deletion of the e3 enhancer, tandem U6-promoter-sgRNA and H1-promoter-sgRNA cassettes were cloned into lentiCRISPR_v2 (Addgene #60954) for single vector expression of two sgRNAs as follows: 1) U6-sgRNA and H1-sgRNA products were generated by PCR amplification using the primers listed in , 2) PCR products were then digested with BsmBI to generate compatible sticky ends, 3) finally, three-way ligation of the two PCR products and BsmBI-digested lentiCRISPR_v2 was performed using T7 DNA ligase (NEB #M0318). .. Control ‘empty’ lentiCRISPR_v2 lacking expression of any sgRNAs was generated by BsmBI digestion, followed by blunting of ends (NEB #E1201) and ligation with T4 DNA ligase (NEB #M0202).

    Article Title: Actomyosin regulation by Eph receptor signaling couples boundary cell formation to border sharpness
    Article Snippet: .. To introduce the K658M mutation in the kinase domain of EphA4a, sgRNA and Cas9 protein were co-injected with a 74 bp donor oligonucleotide (AAGATGCCTGGAAA GCGTGAaATtTGcGTGGCCATAAAAACCCTAAtGGCAGGgTACACCGACAAGCAAAGGCG) containing three silent mutations at the gRNA target site, the K658M mutation and an additional silent mutation that generated an RsaI restriction site. .. Mutations were identified by amplicon restriction using restriction enzymes or T7 endonuclease I (#M0302L NEB) and verified by sequencing.

    other:

    Article Title: One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing
    Article Snippet: For each shot, the equivalent of 4 or 8 µg Cas9 protein (for multiple targets, the total amount was split equally between the different RNPs) in a total volume of 8 µl Cas9 reaction buffer (NEB, B0386A) was mixed with 10 μl gold nanoparticles (3 mg, 0.6 μm in diameter, Bio-Rad) washed twice with Cas9 reaction buffer.

    Construct:

    Article Title: Gene essentiality landscape and druggable oncogenic dependencies in herpesviral primary effusion lymphoma
    Article Snippet: .. Cloning of individual sgRNA constructs All sgRNAs were cloned into plentiGuide-Puro using the BsmBI cloning site (New England Biolabs, Ipswich, MA). sgRNA sequences targeting specific genes were designed using publicly available tools , or were derived from the Brunello library. sgRNA sequences and sequences of primers used for cloning are listed in Supplementary Data . .. Lentivirus preparation For library production, 107 HEK-293T cells were seeded per 15 cm dish the day before transfection.

    Expressing:

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers
    Article Snippet: .. For deletion of the e3 enhancer, tandem U6-promoter-sgRNA and H1-promoter-sgRNA cassettes were cloned into lentiCRISPR_v2 (Addgene #60954) for single vector expression of two sgRNAs as follows: 1) U6-sgRNA and H1-sgRNA products were generated by PCR amplification using the primers listed in , 2) PCR products were then digested with BsmBI to generate compatible sticky ends, 3) finally, three-way ligation of the two PCR products and BsmBI-digested lentiCRISPR_v2 was performed using T7 DNA ligase (NEB #M0318). .. Control ‘empty’ lentiCRISPR_v2 lacking expression of any sgRNAs was generated by BsmBI digestion, followed by blunting of ends (NEB #E1201) and ligation with T4 DNA ligase (NEB #M0202).

    Polymerase Chain Reaction:

    Article Title: Programmed Death Receptor 1 (PD1) Knockout and Human Telomerase Reverse Transcriptase (hTERT) Transduction Can Enhance Persistence and Antitumor Efficacy of Cytokine-Induced Killer Cells Against Hepatocellular Carcinoma
    Article Snippet: .. After the i n vitro transcription template of T7-sgRNAs was amplified by PCR, the sgRNAs were transcribed in vitro using a HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, US). .. The in vitro transcription single-guide RNAs (IVT sgRNAs) were purified by using RNA clean & concentratorTM-25 (Zymo Research, US), eluted in RNase-free water, and used immediately after elution or stored at −80°C.

    Article Title: CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii
    Article Snippet: .. For in vitro assays, 500 ng of sgRNA and 600 ng of Cas9 protein were pre-mixed at 37 °C for 5 min. For experiments, 100 ng of PCR amplicons and 3.1 NEB buffer (NEB, USA) were added, and the mixtures were incubated at 37 °C for 2 hours. .. RNaseA (Bioneer, Republic of Korea) was added, and the mixture was incubated at 37 °C for 20 min. 6× STOP solution (0.5 M EDTA, 80% glycerol, and 10% SDS) was added, and the mixture was incubated 37 °C for 20 min.

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers
    Article Snippet: .. For deletion of the e3 enhancer, tandem U6-promoter-sgRNA and H1-promoter-sgRNA cassettes were cloned into lentiCRISPR_v2 (Addgene #60954) for single vector expression of two sgRNAs as follows: 1) U6-sgRNA and H1-sgRNA products were generated by PCR amplification using the primers listed in , 2) PCR products were then digested with BsmBI to generate compatible sticky ends, 3) finally, three-way ligation of the two PCR products and BsmBI-digested lentiCRISPR_v2 was performed using T7 DNA ligase (NEB #M0318). .. Control ‘empty’ lentiCRISPR_v2 lacking expression of any sgRNAs was generated by BsmBI digestion, followed by blunting of ends (NEB #E1201) and ligation with T4 DNA ligase (NEB #M0202).

    Derivative Assay:

    Article Title: Gene essentiality landscape and druggable oncogenic dependencies in herpesviral primary effusion lymphoma
    Article Snippet: .. Cloning of individual sgRNA constructs All sgRNAs were cloned into plentiGuide-Puro using the BsmBI cloning site (New England Biolabs, Ipswich, MA). sgRNA sequences targeting specific genes were designed using publicly available tools , or were derived from the Brunello library. sgRNA sequences and sequences of primers used for cloning are listed in Supplementary Data . .. Lentivirus preparation For library production, 107 HEK-293T cells were seeded per 15 cm dish the day before transfection.

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    New England Biolabs 1x neb3 buffer
    1x Neb3 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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