bsaji  (New England Biolabs)


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    New England Biolabs bsaji
    Rapidly selecting most effective sgRNAs by single blastocyst genotyping. ( A ) Overview and timeline of the experiment. Beginning wit h experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. ( B ) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing <t>PCR</t> product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by <t>BsaJI,</t> HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.
    Bsaji, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsaji/product/New England Biolabs
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    bsaji - by Bioz Stars, 2022-09
    92/100 stars

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    1) Product Images from "Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs"

    Article Title: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

    Journal: Scientific Reports

    doi: 10.1038/srep13348

    Rapidly selecting most effective sgRNAs by single blastocyst genotyping. ( A ) Overview and timeline of the experiment. Beginning wit h experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. ( B ) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.
    Figure Legend Snippet: Rapidly selecting most effective sgRNAs by single blastocyst genotyping. ( A ) Overview and timeline of the experiment. Beginning wit h experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. ( B ) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.

    Techniques Used: Mutagenesis, Derivative Assay, Injection, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, RFLP Assay, Labeling

    Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis. Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.
    Figure Legend Snippet: Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis. Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.

    Techniques Used: Derivative Assay, FACS, Expressing, Fluorescence, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Mutagenesis

    Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA. ( A ) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. ( B ) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. ( C ) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. Deleted bases are marked with colons, and inserted bases are gray. ( D ) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.
    Figure Legend Snippet: Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA. ( A ) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. ( B ) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. ( C ) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. Deleted bases are marked with colons, and inserted bases are gray. ( D ) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.

    Techniques Used: Knock-Out, Injection, Mutagenesis, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Derivative Assay, Sequencing, Labeling, Western Blot, Expressing

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    New England Biolabs bsaji restriction enzymes
    Confirmation of mutagenesis of targeted loci in representative Enrei-T 1 plants by CAPS analysis. The schematic diagrams above panels show <t>DdeI</t> or <t>BsaJI</t> restriction sites (shaded in gray) in fragments amplified with specific primers. Red and blue nucleotide sequences have the same meaning as those in Fig. 1 . Gray arrows, the sizes of expected wild-type fragments; black arrows, the sizes of expected mutant-type fragments; open triangle, a fragment of unexpected size considered as mutant type. M, molecular weight marker (100-bp ladder)
    Bsaji Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Confirmation of mutagenesis of targeted loci in representative Enrei-T 1 plants by CAPS analysis. The schematic diagrams above panels show DdeI or BsaJI restriction sites (shaded in gray) in fragments amplified with specific primers. Red and blue nucleotide sequences have the same meaning as those in Fig. 1 . Gray arrows, the sizes of expected wild-type fragments; black arrows, the sizes of expected mutant-type fragments; open triangle, a fragment of unexpected size considered as mutant type. M, molecular weight marker (100-bp ladder)

    Journal: BMC Plant Biology

    Article Title: Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis

    doi: 10.1186/s12870-020-02708-6

    Figure Lengend Snippet: Confirmation of mutagenesis of targeted loci in representative Enrei-T 1 plants by CAPS analysis. The schematic diagrams above panels show DdeI or BsaJI restriction sites (shaded in gray) in fragments amplified with specific primers. Red and blue nucleotide sequences have the same meaning as those in Fig. 1 . Gray arrows, the sizes of expected wild-type fragments; black arrows, the sizes of expected mutant-type fragments; open triangle, a fragment of unexpected size considered as mutant type. M, molecular weight marker (100-bp ladder)

    Article Snippet: The PCR was performed under the following conditions: 30 cycles of 94 °C for 30 s, 54 °C (the Gly m Bd 28 K ) or 60 °C (the Gly m Bd 30 K) for 30 s and 72 °C for 60 s. The amplified products were digested with the DdeI and BsaJI restriction enzymes (NEB), respectively, and separated by electrophoresis in 2.0% agarose gels.

    Techniques: Mutagenesis, Amplification, Molecular Weight, Marker

    Rapidly selecting most effective sgRNAs by single blastocyst genotyping. ( A ) Overview and timeline of the experiment. Beginning wit h experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. ( B ) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.

    Journal: Scientific Reports

    Article Title: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

    doi: 10.1038/srep13348

    Figure Lengend Snippet: Rapidly selecting most effective sgRNAs by single blastocyst genotyping. ( A ) Overview and timeline of the experiment. Beginning wit h experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. ( B ) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.

    Article Snippet: Ten microliters of PCR product was digested with BsaJI, HpyCH4V or DraI (New England Biolabs, US), respectively.

    Techniques: Mutagenesis, Derivative Assay, Injection, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, RFLP Assay, Labeling

    Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis. Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.

    Journal: Scientific Reports

    Article Title: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

    doi: 10.1038/srep13348

    Figure Lengend Snippet: Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis. Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.

    Article Snippet: Ten microliters of PCR product was digested with BsaJI, HpyCH4V or DraI (New England Biolabs, US), respectively.

    Techniques: Derivative Assay, FACS, Expressing, Fluorescence, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Mutagenesis

    Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA. ( A ) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. ( B ) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. ( C ) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. Deleted bases are marked with colons, and inserted bases are gray. ( D ) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.

    Journal: Scientific Reports

    Article Title: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

    doi: 10.1038/srep13348

    Figure Lengend Snippet: Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA. ( A ) Newborn wild type pig (upper panel) and piglets carrying Mitf gene mutation (bottom panel). The photographs were taken by the author, Xianlong Wang. ( B ) RFLP agarose gel electrophoresis showing PCR product of target region derived from two piglets digested with BsaJI restriction enzymes. ( C ) Sanger sequencing of the targeting site in mutant pigs. The wild-type (WT) sequence is shown at the top, where the sgRNA sequence is labeled in red and the PAM sequence in purple. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. Deleted bases are marked with colons, and inserted bases are gray. ( D ) Western blot for MITF protein expression in the skin tissues of tail from wild type and mutant piglets. GAPDH was used as a loading control.

    Article Snippet: Ten microliters of PCR product was digested with BsaJI, HpyCH4V or DraI (New England Biolabs, US), respectively.

    Techniques: Knock-Out, Injection, Mutagenesis, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Derivative Assay, Sequencing, Labeling, Western Blot, Expressing

    A) Pedigree of family with clinical features of Snyder-Robinson syndrome. B) DNA electropherograms of the proband (II-2) and a normal male. The normal V132 residue is indicated in red and the G132 mutation residue is indicated in blue. C) BsaJ I digestion

    Journal: American journal of medical genetics. Part A

    Article Title: A missense mutation, p.V132G, in the X-linked spermine synthase gene (SMS) causes Snyder-Robinson syndrome

    doi: 10.1002/ajmg.a.32641

    Figure Lengend Snippet: A) Pedigree of family with clinical features of Snyder-Robinson syndrome. B) DNA electropherograms of the proband (II-2) and a normal male. The normal V132 residue is indicated in red and the G132 mutation residue is indicated in blue. C) BsaJ I digestion

    Article Snippet: 10μl of the amplified PCR product was digested with BsaJ I ( New England Biolabs) and incubated at 60°C.

    Techniques: Mutagenesis