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
    		Buy from Supplier

    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, 2021-04
    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: 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: Inheritance of Hetero-Diploid Pollen S-Haplotype in Self-Compatible Tetraploid Chinese Cherry (Prunus pseudocerasus Lindl)
    Article Snippet: In the present study, the cDNA amplification products using two primer pairs had only one band, but contained several different S-RNase or SFB alleles. .. CSP6I (Invitrogen) was used to digest PCR products of stylar cDNA, and CSP6I (Invitrogen), DpnII, HpyCH4III, HpyCH4IV, BslI and BsaJI (NEB, Mass., USA) were used to digest PCR products of pollen cDNA. ..

    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: 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).

    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: 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. ..

    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. ..

    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.

    Article Title: The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase
    Article Snippet: .. The resulting double-stranded ODNs (7 pmoles) were incubated with 21 pmoles MGMT in a total volume of 17 µl of IBSA (Buffer I containing 0.1% BSA) at room temperature for 3 h and then subjected to digestion with NlaIII, CviKI, StyI, BsaJI or PstI in the buffer provided by the manufacturer (New England Biolabs) in a total volume of 10 µl at room temperature for 1 h. Non-denaturing PAGE gel-loading buffer (30% glycerol, 0.25% xylene cyanol, 0.25% bromophenol blue: 2 µl) was added to each sample, which was then applied to a 20% PAGE gel and subjected to electrophoresis at 100 volts for 1 h. The gels were scanned on a Pharos phosphorimager (BioRad) using the fluorescent gel scanner settings. .. Inhibition of MGMT by O6 -alkG-containing ODNs Duplex ODNs containing O 6 -MeG and O 6 -CMG, but not the control ODN (which contained G in place of the alkylated base), potently inhibited the action of MGMT on [3 H]-methylated substrate DNA.

    Incubation:

    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. ..

    Article Title: The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase
    Article Snippet: .. The resulting double-stranded ODNs (7 pmoles) were incubated with 21 pmoles MGMT in a total volume of 17 µl of IBSA (Buffer I containing 0.1% BSA) at room temperature for 3 h and then subjected to digestion with NlaIII, CviKI, StyI, BsaJI or PstI in the buffer provided by the manufacturer (New England Biolabs) in a total volume of 10 µl at room temperature for 1 h. Non-denaturing PAGE gel-loading buffer (30% glycerol, 0.25% xylene cyanol, 0.25% bromophenol blue: 2 µl) was added to each sample, which was then applied to a 20% PAGE gel and subjected to electrophoresis at 100 volts for 1 h. The gels were scanned on a Pharos phosphorimager (BioRad) using the fluorescent gel scanner settings. .. Inhibition of MGMT by O6 -alkG-containing ODNs Duplex ODNs containing O 6 -MeG and O 6 -CMG, but not the control ODN (which contained G in place of the alkylated base), potently inhibited the action of MGMT on [3 H]-methylated substrate DNA.

    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. ..

    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. ..

    Polyacrylamide Gel Electrophoresis:

    Article Title: The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase
    Article Snippet: .. The resulting double-stranded ODNs (7 pmoles) were incubated with 21 pmoles MGMT in a total volume of 17 µl of IBSA (Buffer I containing 0.1% BSA) at room temperature for 3 h and then subjected to digestion with NlaIII, CviKI, StyI, BsaJI or PstI in the buffer provided by the manufacturer (New England Biolabs) in a total volume of 10 µl at room temperature for 1 h. Non-denaturing PAGE gel-loading buffer (30% glycerol, 0.25% xylene cyanol, 0.25% bromophenol blue: 2 µl) was added to each sample, which was then applied to a 20% PAGE gel and subjected to electrophoresis at 100 volts for 1 h. The gels were scanned on a Pharos phosphorimager (BioRad) using the fluorescent gel scanner settings. .. Inhibition of MGMT by O6 -alkG-containing ODNs Duplex ODNs containing O 6 -MeG and O 6 -CMG, but not the control ODN (which contained G in place of the alkylated base), potently inhibited the action of MGMT on [3 H]-methylated substrate DNA.

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    • restriction endonuclease bsaji  (New England Biolabs)
    99
    New England Biolabs restriction endonuclease bsaji
    Identification of Cacna1f mutations in the nob9 mouse. (A) Sequence analysis of Cacna1f DNA from the nob9 mouse and WT control. The sequence variants for the nob9 involved both a single nucleotide G to A transition (in red) and a 10-nucleotide insertion (in green) within intron 13–14. (B) <t>PCR</t> products produced by <t>BsaJI</t> digestion confirming the G mutation as shown in (A). As a restriction endonuclease, BsaJI is able to recognize and cleave DNA at “CCNNGG” sites.
    Restriction Endonuclease Bsaji, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction endonuclease bsaji/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    restriction endonuclease bsaji - by Bioz Stars, 2021-04
    99/100 stars
    		  Buy from Supplier
    bsaji restriction enzymes  (New England Biolabs)
    93
    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: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsaji restriction enzymes/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsaji restriction enzymes - by Bioz Stars, 2021-04
    93/100 stars
    		  Buy from Supplier

    Image Search Results


    Identification of Cacna1f mutations in the nob9 mouse. (A) Sequence analysis of Cacna1f DNA from the nob9 mouse and WT control. The sequence variants for the nob9 involved both a single nucleotide G to A transition (in red) and a 10-nucleotide insertion (in green) within intron 13–14. (B) PCR products produced by BsaJI digestion confirming the G mutation as shown in (A). As a restriction endonuclease, BsaJI is able to recognize and cleave DNA at “CCNNGG” sites.

    Journal: Experimental eye research

    Article Title: Photoreceptor degeneration in a new Cacna1f mutant mouse model

    doi: 10.1016/j.exer.2018.11.010

    Figure Lengend Snippet: Identification of Cacna1f mutations in the nob9 mouse. (A) Sequence analysis of Cacna1f DNA from the nob9 mouse and WT control. The sequence variants for the nob9 involved both a single nucleotide G to A transition (in red) and a 10-nucleotide insertion (in green) within intron 13–14. (B) PCR products produced by BsaJI digestion confirming the G mutation as shown in (A). As a restriction endonuclease, BsaJI is able to recognize and cleave DNA at “CCNNGG” sites.

    Article Snippet: One of the alterations was detected in a single nucleotide G to A transition near the end of intron 13–14 , and this mutation was further confirmed by PCR products digested by the restriction endonuclease BsaJI.

    Techniques: Sequencing, Polymerase Chain Reaction, Produced, 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

    Results of PCR-RFLP: (a) PCR-amplified target fragment − M = 100-bp marker ladder; well 1 = negative control; 2 = positive control (GADPH primer); 3–5 = samples with 225-bp PCR product and (b) fragments after BsaJI enzyme cutting − M = 50-bp marker ladder, wells 1 and 4 = GG; 2–3 = GT; 5 = TT. PCR-RFLP: polymerase chain reaction–restriction fragment length polymorphism; PCR: polymerase chain reaction; GADPH: glyceraldehyde 3-phosphate dehydrogenase.

    Journal: SAGE Open Medicine

    Article Title: Gene promoter polymorphism of RUNX2 and risk of osteoporosis in postmenopausal Indonesian women

    doi: 10.1177/2050312114531571

    Figure Lengend Snippet: Results of PCR-RFLP: (a) PCR-amplified target fragment − M = 100-bp marker ladder; well 1 = negative control; 2 = positive control (GADPH primer); 3–5 = samples with 225-bp PCR product and (b) fragments after BsaJI enzyme cutting − M = 50-bp marker ladder, wells 1 and 4 = GG; 2–3 = GT; 5 = TT. PCR-RFLP: polymerase chain reaction–restriction fragment length polymorphism; PCR: polymerase chain reaction; GADPH: glyceraldehyde 3-phosphate dehydrogenase.

    Article Snippet: Restriction fragment length polymorphism (RFLP) analysis was performed by inserting the restriction enzyme BsaJI (New England Biolabs (NEB), 1 µL, 10U/µL) into a tube containing the amplified DNA fragments, 2 µL RE10X buffer solution and 18 µL ddH2 O.

    Techniques: Polymerase Chain Reaction, Amplification, Marker, Negative Control, Positive Control

    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