bsaji  (New England Biolabs)


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    Name:
    BsaJI
    Description:
    BsaJI 5 000 units
    Catalog Number:
    r0536l
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    Structured Review

    New England Biolabs bsaji
    BsaJI
    BsaJI 5 000 units
    https://www.bioz.com/result/bsaji/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsaji - by Bioz Stars, 2021-03
    93/100 stars

    Images

    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

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs
    Article Snippet: The identity of each PCR product was confirmed by restriction fragment length polymorphism (RFLP) analysis. .. Ten microliters of PCR product was digested with BsaJI, HpyCH4V or DraI (New England Biolabs, US), respectively. .. The reaction products were separated by 2% agarose gel electrophoresis in the presence of ethidium bromide solution, and visualized with a UV transilluminator (UVP, Upland, CA).

    Article Title: Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis
    Article Snippet: The targeted regions in the Gly m Bd 28 K and Gly m Bd 30 K loci were amplified by PCR with specific primers (Additional file : Table S3). .. 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. .. The DNA fragments of expected digested-pattern derived from the targeted region carrying mutations and those with no mutations were considered as the mutant type and wild type, respectively.

    Article Title: A missense mutation, p.V132G, in the X-linked spermine synthase gene (SMS) causes Snyder-Robinson syndrome
    Article Snippet: .. 10μl of the amplified PCR product was digested with BsaJ I ( New England Biolabs) and incubated at 60°C. ..

    Article Title: Two single nucleotide polymorphisms in the von Hippel-Lindau tumor suppressor gene in Taiwanese with renal cell carcinoma
    Article Snippet: PCR conditions included initial denaturation for 5 min at 95°C, followed by 35 cycles consisting of 1 min at 95°C, 1 min at 60°C for rs779805 or 50°C for rs1642742, and 2 min at 72°C, with final extension for 10 min at 72°C. .. Digestion of restriction enzyme An aliquot of 10 µl of PCR product was digested with 2.5 units BsaJ I (New England Biolabs) for rs779805 or 2.5 units Acc I (New England Biolabs) for rs1642742 in a total volume of 20 µl that contained one-fold NEBuffer 2 or NEBuffer 4. ..

    Formalin-fixed Paraffin-Embedded:

    Article Title: Quantitative and Sensitive Detection of Cancer Genome Amplifications from Formalin Fixed Paraffin Embedded Tumors with Droplet Digital PCR
    Article Snippet: The digestion step was not required for FFPE samples as the DNA tends to be highly fragmented, but was performed here to maintain experimental consistency. .. To assess FGFR2 copy number, 125 ng of each FFPE sample was digested with 1.25 units of BsaJI (New England BioLabs) in 15 μL for 1 h at 60°C. ..

    Amplification:

    Article Title: Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis
    Article Snippet: The targeted regions in the Gly m Bd 28 K and Gly m Bd 30 K loci were amplified by PCR with specific primers (Additional file : Table S3). .. 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. .. The DNA fragments of expected digested-pattern derived from the targeted region carrying mutations and those with no mutations were considered as the mutant type and wild type, respectively.

    Article Title: A missense mutation, p.V132G, in the X-linked spermine synthase gene (SMS) causes Snyder-Robinson syndrome
    Article Snippet: .. 10μl of the amplified PCR product was digested with BsaJ I ( New England Biolabs) and incubated at 60°C. ..

    Article Title: Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies
    Article Snippet: If needed to obtain optimal signal intensities, the exposure times were sometimes varied, as judged by evaluation of the hybridization controls present on each membrane. .. Restriction Fragment Length Polymorphism (RFLP) After TP53 exon 4 amplification, 7 μL of the amplified reaction products were digested in a 10 μL volume of 1 × NEB buffer 2 and 5 U of Bsa JI or Bst UI endonucleases (New England BioLabs, Newton, MA) at 60°C for at least 2 hours. ..

    Article Title: Evidence for association of STAT4 and IL12RB2 variants with Myasthenia gravis susceptibility: What is the effect on gene expression in thymus?
    Article Snippet: Myasthenia gravis (MG) is an autoimmune disease mediated by the presence of autoantibodies that bind mainly to the acetylcholine receptor (AChR) in the neuromuscular junction. .. Myasthenia gravis (MG) is an autoimmune disease mediated by the presence of autoantibodies that bind mainly to the acetylcholine receptor (AChR) in the neuromuscular junction. ..

    Electrophoresis:

    Article Title: Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis
    Article Snippet: The targeted regions in the Gly m Bd 28 K and Gly m Bd 30 K loci were amplified by PCR with specific primers (Additional file : Table S3). .. 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. .. The DNA fragments of expected digested-pattern derived from the targeted region carrying mutations and those with no mutations were considered as the mutant type and wild type, respectively.

    Incubation:

    Article Title: A missense mutation, p.V132G, in the X-linked spermine synthase gene (SMS) causes Snyder-Robinson syndrome
    Article Snippet: .. 10μl of the amplified PCR product was digested with BsaJ I ( New England Biolabs) and incubated at 60°C. ..

    Article Title: Evidence for association of STAT4 and IL12RB2 variants with Myasthenia gravis susceptibility: What is the effect on gene expression in thymus?
    Article Snippet: Myasthenia gravis (MG) is an autoimmune disease mediated by the presence of autoantibodies that bind mainly to the acetylcholine receptor (AChR) in the neuromuscular junction. .. Myasthenia gravis (MG) is an autoimmune disease mediated by the presence of autoantibodies that bind mainly to the acetylcholine receptor (AChR) in the neuromuscular junction. ..

    Agarose Gel Electrophoresis:

    Article Title: Evidence for association of STAT4 and IL12RB2 variants with Myasthenia gravis susceptibility: What is the effect on gene expression in thymus?
    Article Snippet: Myasthenia gravis (MG) is an autoimmune disease mediated by the presence of autoantibodies that bind mainly to the acetylcholine receptor (AChR) in the neuromuscular junction. .. Myasthenia gravis (MG) is an autoimmune disease mediated by the presence of autoantibodies that bind mainly to the acetylcholine receptor (AChR) in the neuromuscular junction. ..

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  • 85
    New England Biolabs bsa ji
    p53 codon 72 genotyping . (A) PCR for TP53 exon 4 detection. PCR products were loaded on 1.5% agarose gel. 100 base pairs DNA ladder (lane 1), positive controls (lane 2 and 3), negative controls (lanes 4 and 26), samples tested (lanes 5-25). (B) Detection of TP53 codon 72 polymorphism by DHPLC. Elution profiles obtained at 59°C. Representative heterozygous (a) and homozygous profile (b and c) are depicted. Note the small shoulder only in heterozygous samples. After the first trial, homozygous samples were mixed in approximately equimolar proportions with a control sample with TP53 codon 72 polymorphism previously identified as homozygous proline. This allowed differentiating the two homozygous genotypes. Homozygous profiles represent a sample with the genotype similar to the control sample added. Heterozygous profile represents a sample with the genotype differing from the control sample. (C) Dot Blot hybridization for TP53 codon 72 polymorphism genotyping. Samples were spotted onto nylon membranes and hybridized with biotin-labelled oligonucleotide probes for allele Arg (PArg) and allele Pro (PPro). Sample 1A, 1B and 2A represent positive controls. The profile observed in sample 1A is compatible with a homozygous arginine. Sample 1B presents a profile compatible with the genotype homozygous proline. Sample 2A presents a profile compatible with the heterozygous genotype. (D) Silver-stained 8% polyacrylamide gel showing the restriction profiles obtained with enzyme <t>Bst</t> UI of the TP53 exon 4 PCR product. 50 base pairs DNA ladder (lane 1). Lanes 2, 4, 5 represent heterozygous samples where fragments of 279, 160 and 119 base pairs can be detected. Lanes 3 and 8 represent homozygous proline samples in which the enzyme was not able to digest the PCR product. Lanes 6 represent an homozygous arginine in which fragments of 160 and 119 base pairs can be identified. Lanes 7, 9 and 10 represent positive controls for homozygous arginine, homozygous proline and heterozygous samples, respectively. (E) Silver-stained 12% polyacrylamide gel showing the restriction profiles obtained with enzyme <t>Bsa</t> JI of the TP53 exon 4 PCR product. 50 base pairs DNA ladder (lane 1). Lanes 3, 4, 5 represent heterozygous samples in which fragments of 138, 94, 66, 44, 41 and 34 base pairs can be detected. Lanes 7 represent a homozygous proline sample in which fragments of 94, 66, 44, 41 and 34 base pairs can be identified. Lanes 2, 6, 8, 10 and 11 represent homozygous arginine samples in which fragments of 138, 66, 41 and 34 base pairs can be detected. Lanes 9, 12 and 13 represent positive controls for heterozygous, homozygous arginine and homozygous proline samples, respectively.
    Bsa Ji, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsa ji/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsa ji - by Bioz Stars, 2021-03
    85/100 stars
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    Image Search Results


    p53 codon 72 genotyping . (A) PCR for TP53 exon 4 detection. PCR products were loaded on 1.5% agarose gel. 100 base pairs DNA ladder (lane 1), positive controls (lane 2 and 3), negative controls (lanes 4 and 26), samples tested (lanes 5-25). (B) Detection of TP53 codon 72 polymorphism by DHPLC. Elution profiles obtained at 59°C. Representative heterozygous (a) and homozygous profile (b and c) are depicted. Note the small shoulder only in heterozygous samples. After the first trial, homozygous samples were mixed in approximately equimolar proportions with a control sample with TP53 codon 72 polymorphism previously identified as homozygous proline. This allowed differentiating the two homozygous genotypes. Homozygous profiles represent a sample with the genotype similar to the control sample added. Heterozygous profile represents a sample with the genotype differing from the control sample. (C) Dot Blot hybridization for TP53 codon 72 polymorphism genotyping. Samples were spotted onto nylon membranes and hybridized with biotin-labelled oligonucleotide probes for allele Arg (PArg) and allele Pro (PPro). Sample 1A, 1B and 2A represent positive controls. The profile observed in sample 1A is compatible with a homozygous arginine. Sample 1B presents a profile compatible with the genotype homozygous proline. Sample 2A presents a profile compatible with the heterozygous genotype. (D) Silver-stained 8% polyacrylamide gel showing the restriction profiles obtained with enzyme Bst UI of the TP53 exon 4 PCR product. 50 base pairs DNA ladder (lane 1). Lanes 2, 4, 5 represent heterozygous samples where fragments of 279, 160 and 119 base pairs can be detected. Lanes 3 and 8 represent homozygous proline samples in which the enzyme was not able to digest the PCR product. Lanes 6 represent an homozygous arginine in which fragments of 160 and 119 base pairs can be identified. Lanes 7, 9 and 10 represent positive controls for homozygous arginine, homozygous proline and heterozygous samples, respectively. (E) Silver-stained 12% polyacrylamide gel showing the restriction profiles obtained with enzyme Bsa JI of the TP53 exon 4 PCR product. 50 base pairs DNA ladder (lane 1). Lanes 3, 4, 5 represent heterozygous samples in which fragments of 138, 94, 66, 44, 41 and 34 base pairs can be detected. Lanes 7 represent a homozygous proline sample in which fragments of 94, 66, 44, 41 and 34 base pairs can be identified. Lanes 2, 6, 8, 10 and 11 represent homozygous arginine samples in which fragments of 138, 66, 41 and 34 base pairs can be detected. Lanes 9, 12 and 13 represent positive controls for heterozygous, homozygous arginine and homozygous proline samples, respectively.

    Journal: BMC Genetics

    Article Title: Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies

    doi: 10.1186/1471-2156-11-44

    Figure Lengend Snippet: p53 codon 72 genotyping . (A) PCR for TP53 exon 4 detection. PCR products were loaded on 1.5% agarose gel. 100 base pairs DNA ladder (lane 1), positive controls (lane 2 and 3), negative controls (lanes 4 and 26), samples tested (lanes 5-25). (B) Detection of TP53 codon 72 polymorphism by DHPLC. Elution profiles obtained at 59°C. Representative heterozygous (a) and homozygous profile (b and c) are depicted. Note the small shoulder only in heterozygous samples. After the first trial, homozygous samples were mixed in approximately equimolar proportions with a control sample with TP53 codon 72 polymorphism previously identified as homozygous proline. This allowed differentiating the two homozygous genotypes. Homozygous profiles represent a sample with the genotype similar to the control sample added. Heterozygous profile represents a sample with the genotype differing from the control sample. (C) Dot Blot hybridization for TP53 codon 72 polymorphism genotyping. Samples were spotted onto nylon membranes and hybridized with biotin-labelled oligonucleotide probes for allele Arg (PArg) and allele Pro (PPro). Sample 1A, 1B and 2A represent positive controls. The profile observed in sample 1A is compatible with a homozygous arginine. Sample 1B presents a profile compatible with the genotype homozygous proline. Sample 2A presents a profile compatible with the heterozygous genotype. (D) Silver-stained 8% polyacrylamide gel showing the restriction profiles obtained with enzyme Bst UI of the TP53 exon 4 PCR product. 50 base pairs DNA ladder (lane 1). Lanes 2, 4, 5 represent heterozygous samples where fragments of 279, 160 and 119 base pairs can be detected. Lanes 3 and 8 represent homozygous proline samples in which the enzyme was not able to digest the PCR product. Lanes 6 represent an homozygous arginine in which fragments of 160 and 119 base pairs can be identified. Lanes 7, 9 and 10 represent positive controls for homozygous arginine, homozygous proline and heterozygous samples, respectively. (E) Silver-stained 12% polyacrylamide gel showing the restriction profiles obtained with enzyme Bsa JI of the TP53 exon 4 PCR product. 50 base pairs DNA ladder (lane 1). Lanes 3, 4, 5 represent heterozygous samples in which fragments of 138, 94, 66, 44, 41 and 34 base pairs can be detected. Lanes 7 represent a homozygous proline sample in which fragments of 94, 66, 44, 41 and 34 base pairs can be identified. Lanes 2, 6, 8, 10 and 11 represent homozygous arginine samples in which fragments of 138, 66, 41 and 34 base pairs can be detected. Lanes 9, 12 and 13 represent positive controls for heterozygous, homozygous arginine and homozygous proline samples, respectively.

    Article Snippet: Restriction Fragment Length Polymorphism (RFLP) After TP53 exon 4 amplification, 7 μL of the amplified reaction products were digested in a 10 μL volume of 1 × NEB buffer 2 and 5 U of Bsa JI or Bst UI endonucleases (New England BioLabs, Newton, MA) at 60°C for at least 2 hours.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Dot Blot, Hybridization, Staining

    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

    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