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    Name:
    ScaI HF
    Description:
    ScaI HF 5 000 units
    Catalog Number:
    r3122l
    Price:
    269
    Size:
    5 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs scai hf
    ScaI HF
    ScaI HF 5 000 units
    https://www.bioz.com/result/scai hf/product/New England Biolabs
    Average 96 stars, based on 20937 article reviews
    Price from $9.99 to $1999.99
    scai hf - by Bioz Stars, 2020-08
    96/100 stars

    Images

    1) Product Images from "The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair"

    Article Title: The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz834

    Effect of dCas9 roadblock on incision of GATC sites during initiation of DNA mismatch repair. ( A ) DNA substrates contain a single G/T mismatch and Alexa 647 fluorophore at different positions flanked by two hemi-methylated GATC sites and are linearized using ScaI. ( B ) Time courses for strand incision by 50 nM MutS, 50 nM MutL and 25 nM MutH on 0.5 nM GT#2 (left panel; substrate carrying mismatch) or AT#2 (right panel; control containing the corresponding A/T Watson-Crick base pair). Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using Alexa 647 fluorophore emission. ( C ) Schematic of the binding and disassociation of dCas9 RNP complexes on target DNA during surface plasmon resonance analysis using Biacore T100. ( D ) Left panel: dCas9-RNA_A complex (0.6–20 nM) injected over 60-bp duplex DNA carrying target site C. Right panel: dCas9-RNA_C complex (0.6–20 nM) injected over 60-bp duplex DNA carrying site (C). ( E ) Control addressing putative rebinding of dCas9-RNP to target site during dissociation. dCas9-RNA_C (20 nM) was injected over the surface, and dissociation was monitored in the absence (dark orange) or presence (blue) of excess non-biotinylated duplex containing target site C (competitor). As control, dCas9-RNA_C was mixed with excess competitor before being injected over the surface (light orange). (F, G) Time courses for hemimethylated GATC site incision on 0.5 nM GT#2 ( F ) and 0.5 nM GT#2b ( G ) by 50 nM MutS, 50 nM MutL and 25 nM MutH in the absence (left) and presence (right) of a dCas9 roadblock placed on target site C in between the GATC sites. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs underneath the gels show fractions of product containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments.
    Figure Legend Snippet: Effect of dCas9 roadblock on incision of GATC sites during initiation of DNA mismatch repair. ( A ) DNA substrates contain a single G/T mismatch and Alexa 647 fluorophore at different positions flanked by two hemi-methylated GATC sites and are linearized using ScaI. ( B ) Time courses for strand incision by 50 nM MutS, 50 nM MutL and 25 nM MutH on 0.5 nM GT#2 (left panel; substrate carrying mismatch) or AT#2 (right panel; control containing the corresponding A/T Watson-Crick base pair). Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using Alexa 647 fluorophore emission. ( C ) Schematic of the binding and disassociation of dCas9 RNP complexes on target DNA during surface plasmon resonance analysis using Biacore T100. ( D ) Left panel: dCas9-RNA_A complex (0.6–20 nM) injected over 60-bp duplex DNA carrying target site C. Right panel: dCas9-RNA_C complex (0.6–20 nM) injected over 60-bp duplex DNA carrying site (C). ( E ) Control addressing putative rebinding of dCas9-RNP to target site during dissociation. dCas9-RNA_C (20 nM) was injected over the surface, and dissociation was monitored in the absence (dark orange) or presence (blue) of excess non-biotinylated duplex containing target site C (competitor). As control, dCas9-RNA_C was mixed with excess competitor before being injected over the surface (light orange). (F, G) Time courses for hemimethylated GATC site incision on 0.5 nM GT#2 ( F ) and 0.5 nM GT#2b ( G ) by 50 nM MutS, 50 nM MutL and 25 nM MutH in the absence (left) and presence (right) of a dCas9 roadblock placed on target site C in between the GATC sites. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs underneath the gels show fractions of product containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments.

    Techniques Used: Methylation, Nucleic Acid Electrophoresis, Binding Assay, SPR Assay, Injection

    2) Product Images from "The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair"

    Article Title: The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz834

    Effect of dCas9 roadblock on incision of GATC sites during initiation of DNA mismatch repair. ( A ) DNA substrates contain a single G/T mismatch and Alexa 647 fluorophore at different positions flanked by two hemi-methylated GATC sites and are linearized using ScaI. ( B ) Time courses for strand incision by 50 nM MutS, 50 nM MutL and 25 nM MutH on 0.5 nM GT#2 (left panel; substrate carrying mismatch) or AT#2 (right panel; control containing the corresponding A/T Watson-Crick base pair). Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using Alexa 647 fluorophore emission. ( C ) Schematic of the binding and disassociation of dCas9 RNP complexes on target DNA during surface plasmon resonance analysis using Biacore T100. ( D ) Left panel: dCas9-RNA_A complex (0.6–20 nM) injected over 60-bp duplex DNA carrying target site C. Right panel: dCas9-RNA_C complex (0.6–20 nM) injected over 60-bp duplex DNA carrying site (C). ( E ) Control addressing putative rebinding of dCas9-RNP to target site during dissociation. dCas9-RNA_C (20 nM) was injected over the surface, and dissociation was monitored in the absence (dark orange) or presence (blue) of excess non-biotinylated duplex containing target site C (competitor). As control, dCas9-RNA_C was mixed with excess competitor before being injected over the surface (light orange). (F, G) Time courses for hemimethylated GATC site incision on 0.5 nM GT#2 ( F ) and 0.5 nM GT#2b ( G ) by 50 nM MutS, 50 nM MutL and 25 nM MutH in the absence (left) and presence (right) of a dCas9 roadblock placed on target site C in between the GATC sites. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs underneath the gels show fractions of product containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments.
    Figure Legend Snippet: Effect of dCas9 roadblock on incision of GATC sites during initiation of DNA mismatch repair. ( A ) DNA substrates contain a single G/T mismatch and Alexa 647 fluorophore at different positions flanked by two hemi-methylated GATC sites and are linearized using ScaI. ( B ) Time courses for strand incision by 50 nM MutS, 50 nM MutL and 25 nM MutH on 0.5 nM GT#2 (left panel; substrate carrying mismatch) or AT#2 (right panel; control containing the corresponding A/T Watson-Crick base pair). Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using Alexa 647 fluorophore emission. ( C ) Schematic of the binding and disassociation of dCas9 RNP complexes on target DNA during surface plasmon resonance analysis using Biacore T100. ( D ) Left panel: dCas9-RNA_A complex (0.6–20 nM) injected over 60-bp duplex DNA carrying target site C. Right panel: dCas9-RNA_C complex (0.6–20 nM) injected over 60-bp duplex DNA carrying site (C). ( E ) Control addressing putative rebinding of dCas9-RNP to target site during dissociation. dCas9-RNA_C (20 nM) was injected over the surface, and dissociation was monitored in the absence (dark orange) or presence (blue) of excess non-biotinylated duplex containing target site C (competitor). As control, dCas9-RNA_C was mixed with excess competitor before being injected over the surface (light orange). (F, G) Time courses for hemimethylated GATC site incision on 0.5 nM GT#2 ( F ) and 0.5 nM GT#2b ( G ) by 50 nM MutS, 50 nM MutL and 25 nM MutH in the absence (left) and presence (right) of a dCas9 roadblock placed on target site C in between the GATC sites. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs underneath the gels show fractions of product containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments.

    Techniques Used: Methylation, Nucleic Acid Electrophoresis, Binding Assay, SPR Assay, Injection

    3) Product Images from "Mutants of Cre recombinase with improved accuracy"

    Article Title: Mutants of Cre recombinase with improved accuracy

    Journal: Nature communications

    doi: 10.1038/ncomms3509

    In vivo recombination of plasmids by mutants of Cre a) Plasmid architecture of the three expected recombination products. Recombination sites are shown as triangles. For simplicity, the map is in linear form, where the NcoI sites at the ends represent a single NcoI site on the circular plasmids. The numbers under each segment indicate distance in bp (map not drawn to scale). The numbers adjacent to each map are the sizes of the expected digestion products, with the asterisks indicating the product size that is unique to the particular configuration. b) Digest analysis of loxP x loxP (left five lanes) and ψlox h7q21 x ψCore h7q21 (right five lanes) recombination. (c d) Inversion and recombination frequency of (c) loxP x loxP and (d) ψCore h7q21 x ψlox h7q21 recombination obtained by quantifying band intensities. Error bars correspond to 95% C.I. (n= 2 independent experiment). NI: NcoI site; SI: ScaI site.
    Figure Legend Snippet: In vivo recombination of plasmids by mutants of Cre a) Plasmid architecture of the three expected recombination products. Recombination sites are shown as triangles. For simplicity, the map is in linear form, where the NcoI sites at the ends represent a single NcoI site on the circular plasmids. The numbers under each segment indicate distance in bp (map not drawn to scale). The numbers adjacent to each map are the sizes of the expected digestion products, with the asterisks indicating the product size that is unique to the particular configuration. b) Digest analysis of loxP x loxP (left five lanes) and ψlox h7q21 x ψCore h7q21 (right five lanes) recombination. (c d) Inversion and recombination frequency of (c) loxP x loxP and (d) ψCore h7q21 x ψlox h7q21 recombination obtained by quantifying band intensities. Error bars correspond to 95% C.I. (n= 2 independent experiment). NI: NcoI site; SI: ScaI site.

    Techniques Used: In Vivo, Plasmid Preparation

    4) Product Images from "Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans"

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.117.300216

    lgc-35 (L324S) CRISPR-Cas9-RNP genotyping and Mendelian segregation. (A) Top, exon-intron structure of the lgc-35 genomic locus and the PCR strategy used for identifying edited animals. PCR primers were designed to produce a single 820 bp product that flanks the edit of interest. Wild-type alleles (+/+) lacking the edited and ScaI restriction site show a single 820 bp band. Successfully edited heterozygotes (m/+) produce three bands (820, 512, and 308 bp), and homozygotes (m/m) produce two bands (512 and 308 bp) when digested with ScaI. Bottom, genotyping data from the 24 positive red fluorescent progeny chosen from three different P0 injected animals (D, L, I). PCR restriction digest analyses revealed that F1 animals D-2,4,5,7,8, L-2, and I-3 are heterozygous knock-ins. An N2 control is shown at the far right of the gel. PCR failures occurred for animals D-3, L-1,8, and I-6. (B) Representative chromatogram from heterozygous animal D-2. Nucleotide codon spacing and amino acid translations are shown above for the heterozygote and wild-type strands. The desired edit is highlighted by a red box, and the conservative changes that create an in-frame ScaI restriction site are highlighted by a yellow box. (C) Left, Mendelian analysis from animal D-2. 96 single F2 progeny were picked and genotyped. The total for the analysis was 81, since there were 15 PCR failures. Mendelian segregation reveals 17 wild-type (+/+), 46 heterozygote (m/+), and 18 homozygote (m/m) animals. The green outline box shows representative wild type (+/+), the blue outline box shows representative heterozygotes (m/+), and red outline box shows representative homozygotes (m/m). Right, representative chromatogram from a homozygous knock-in animal.
    Figure Legend Snippet: lgc-35 (L324S) CRISPR-Cas9-RNP genotyping and Mendelian segregation. (A) Top, exon-intron structure of the lgc-35 genomic locus and the PCR strategy used for identifying edited animals. PCR primers were designed to produce a single 820 bp product that flanks the edit of interest. Wild-type alleles (+/+) lacking the edited and ScaI restriction site show a single 820 bp band. Successfully edited heterozygotes (m/+) produce three bands (820, 512, and 308 bp), and homozygotes (m/m) produce two bands (512 and 308 bp) when digested with ScaI. Bottom, genotyping data from the 24 positive red fluorescent progeny chosen from three different P0 injected animals (D, L, I). PCR restriction digest analyses revealed that F1 animals D-2,4,5,7,8, L-2, and I-3 are heterozygous knock-ins. An N2 control is shown at the far right of the gel. PCR failures occurred for animals D-3, L-1,8, and I-6. (B) Representative chromatogram from heterozygous animal D-2. Nucleotide codon spacing and amino acid translations are shown above for the heterozygote and wild-type strands. The desired edit is highlighted by a red box, and the conservative changes that create an in-frame ScaI restriction site are highlighted by a yellow box. (C) Left, Mendelian analysis from animal D-2. 96 single F2 progeny were picked and genotyped. The total for the analysis was 81, since there were 15 PCR failures. Mendelian segregation reveals 17 wild-type (+/+), 46 heterozygote (m/+), and 18 homozygote (m/m) animals. The green outline box shows representative wild type (+/+), the blue outline box shows representative heterozygotes (m/+), and red outline box shows representative homozygotes (m/m). Right, representative chromatogram from a homozygous knock-in animal.

    Techniques Used: CRISPR, Polymerase Chain Reaction, Injection, Knock-In

    5) Product Images from "ZO-1 and ZO-2 Are Required for Extra-Embryonic Endoderm Integrity, Primitive Ectoderm Survival and Normal Cavitation in Embryoid Bodies Derived from Mouse Embryonic Stem Cells"

    Article Title: ZO-1 and ZO-2 Are Required for Extra-Embryonic Endoderm Integrity, Primitive Ectoderm Survival and Normal Cavitation in Embryoid Bodies Derived from Mouse Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099532

    Targeting of the ZO-1 and ZO-2 locus and genotyping. ( A ) Targeting strategy. Schematic representation of the genomic loci restriction maps of ZO-1 showing exon 1 (panel a) and ZO-2 showing exons 2–3 (panel b) in yellow boxes with the initiation ATG. Through the in-frame insertion of a LacZ gene (green box) and a loxP-flanked (purple circle) Neo cassette (white box) immediately downstream of the ATG codon, the ZO-1 and ZO-2 allele-targeting constructs were designed to delete the entire ZO-1 exon 1 and part of the downstream intron; and part of ZO-2 exon 2 respectively. The red bar indicates the position of probe hybridization for Southern blot analysis. ( B ) Genotypic analysis by Southern blotting. ScaI-digested genomic DNA of selected mESC clones were hybridized with a DIG-labelled 5′ genomic DNA probe for the identification of homologous recombinants. 10 kb and 14.8 kb probe-hybridized fragments correspond to WT and targeted allele of ZO-1 locus respectively (panel a), whereas WT and targeted allele of ZO-2 locus are respectively represented by 6.7 kb and 11.5 kb fragments (panel b). ( C ) Protein expression analysis. mESC lysates were subjected to immunoblotting with anti-ZO-1 and anti-ZO-2 antibodies. The presence of ZO-1 and ZO-2 protein is indicated by 220 kD and 160 kD bands respectively. GAPDH served as a control for equal lysate input.
    Figure Legend Snippet: Targeting of the ZO-1 and ZO-2 locus and genotyping. ( A ) Targeting strategy. Schematic representation of the genomic loci restriction maps of ZO-1 showing exon 1 (panel a) and ZO-2 showing exons 2–3 (panel b) in yellow boxes with the initiation ATG. Through the in-frame insertion of a LacZ gene (green box) and a loxP-flanked (purple circle) Neo cassette (white box) immediately downstream of the ATG codon, the ZO-1 and ZO-2 allele-targeting constructs were designed to delete the entire ZO-1 exon 1 and part of the downstream intron; and part of ZO-2 exon 2 respectively. The red bar indicates the position of probe hybridization for Southern blot analysis. ( B ) Genotypic analysis by Southern blotting. ScaI-digested genomic DNA of selected mESC clones were hybridized with a DIG-labelled 5′ genomic DNA probe for the identification of homologous recombinants. 10 kb and 14.8 kb probe-hybridized fragments correspond to WT and targeted allele of ZO-1 locus respectively (panel a), whereas WT and targeted allele of ZO-2 locus are respectively represented by 6.7 kb and 11.5 kb fragments (panel b). ( C ) Protein expression analysis. mESC lysates were subjected to immunoblotting with anti-ZO-1 and anti-ZO-2 antibodies. The presence of ZO-1 and ZO-2 protein is indicated by 220 kD and 160 kD bands respectively. GAPDH served as a control for equal lysate input.

    Techniques Used: Construct, Hybridization, Southern Blot, Clone Assay, Expressing

    Related Articles

    Clone Assay:

    Article Title: ZO-1 and ZO-2 Are Required for Extra-Embryonic Endoderm Integrity, Primitive Ectoderm Survival and Normal Cavitation in Embryoid Bodies Derived from Mouse Embryonic Stem Cells
    Article Snippet: .. Southern blot analysis Genomic DNA was extracted from G418-resistant ESC clones and completely restriction digested with ScaI-HF (New England BioLabs). .. For the ZO-1 locus, a 554 bp ZO-1 probe, corresponding to the 5′ arm of the targeted allele, was used to detect the 10 kb and 14.8 kb bands from the WT and mutant alleles respectively.

    Agarose Gel Electrophoresis:

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel. .. Molecular biology The transient fluorescent marker plasmid pCFJ90 (a gift from C. Frojkær-Jensen) contains the C. elegans myo-2 promoter, a worm-optimized mCherry fluorophore, and an unc-54 transcriptional terminator sequence that is specifically expressed in pharyngeal muscle.

    Southern Blot:

    Article Title: ZO-1 and ZO-2 Are Required for Extra-Embryonic Endoderm Integrity, Primitive Ectoderm Survival and Normal Cavitation in Embryoid Bodies Derived from Mouse Embryonic Stem Cells
    Article Snippet: .. Southern blot analysis Genomic DNA was extracted from G418-resistant ESC clones and completely restriction digested with ScaI-HF (New England BioLabs). .. For the ZO-1 locus, a 554 bp ZO-1 probe, corresponding to the 5′ arm of the targeted allele, was used to detect the 10 kb and 14.8 kb bands from the WT and mutant alleles respectively.

    Purification:

    Article Title: Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes
    Article Snippet: .. PCR products were purified using QIAquick purification columns (Qiagen) and digested overnight at 37°C in a 10 μl reaction containing ScaI-HF endonuclease (New England BioLabs) according to the manufacturer’s instructions. ..

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel. .. Molecular biology The transient fluorescent marker plasmid pCFJ90 (a gift from C. Frojkær-Jensen) contains the C. elegans myo-2 promoter, a worm-optimized mCherry fluorophore, and an unc-54 transcriptional terminator sequence that is specifically expressed in pharyngeal muscle.

    Article Title: Mutants of Cre recombinase with improved accuracy
    Article Snippet: .. The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9). .. The digests quantified on 1% agarose gels following purification using the QIAprep Spin Miniprep kit.

    Electrophoresis:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone. .. The appropriate bands corresponding to the TP53 exon 5 insert were excised from the gel and purified using the Zymoclean Gel DNA Recovery kit (Zymo Research) and DNA was quantified via spectrophotometry.

    Polymerase Chain Reaction:

    Article Title: Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes
    Article Snippet: .. PCR products were purified using QIAquick purification columns (Qiagen) and digested overnight at 37°C in a 10 μl reaction containing ScaI-HF endonuclease (New England BioLabs) according to the manufacturer’s instructions. ..

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans
    Article Snippet: .. The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel. .. Molecular biology The transient fluorescent marker plasmid pCFJ90 (a gift from C. Frojkær-Jensen) contains the C. elegans myo-2 promoter, a worm-optimized mCherry fluorophore, and an unc-54 transcriptional terminator sequence that is specifically expressed in pharyngeal muscle.

    other:

    Article Title: The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair
    Article Snippet: The fragment indicative for nicking at GATC site 2 was created by digesting GT#2647 using ScaI-HF and BamHI for 30 min at at 37°C.

    Marker:

    Article Title: The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair
    Article Snippet: .. Marker was created as follows: GT#2647 substrate was linearized with ScaI-HF to create the full-length linear fragment. .. The fragment indicative for nicking at GATC site 1 was created by digesting GT#1b ( ) for 30 min at 37°C with ScaI-HF followed by 30 min at 60°C with BstYI.

    Plasmid Preparation:

    Article Title: Targeted single molecule mutation detection with massively parallel sequencing
    Article Snippet: .. Both mixtures were subjected to double enzymatic digestion using AfeI (New England BioLabs) and ScaI-HF (New England BioLabs) and run on a 1.5% UltraPure Low-Melting Point Agarose (Invitrogen) electrophoresis gel with 1× SYBR Safe (Life Technologies, Grand Island, NY, USA) to separate the TP53 exon 5 insert from the TOPO vector backbone. .. The appropriate bands corresponding to the TP53 exon 5 insert were excised from the gel and purified using the Zymoclean Gel DNA Recovery kit (Zymo Research) and DNA was quantified via spectrophotometry.

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    New England Biolabs scai hf
    Effect of dCas9 roadblock on incision of <t>GATC</t> sites during initiation of DNA mismatch repair. ( A ) DNA substrates contain a single G/T mismatch and Alexa 647 fluorophore at different positions flanked by two hemi-methylated GATC sites and are linearized using <t>ScaI.</t> ( B ) Time courses for strand incision by 50 nM MutS, 50 nM MutL and 25 nM MutH on 0.5 nM GT#2 (left panel; substrate carrying mismatch) or AT#2 (right panel; control containing the corresponding A/T Watson-Crick base pair). Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using Alexa 647 fluorophore emission. ( C ) Schematic of the binding and disassociation of dCas9 RNP complexes on target DNA during surface plasmon resonance analysis using Biacore T100. ( D ) Left panel: dCas9-RNA_A complex (0.6–20 nM) injected over 60-bp duplex DNA carrying target site C. Right panel: dCas9-RNA_C complex (0.6–20 nM) injected over 60-bp duplex DNA carrying site (C). ( E ) Control addressing putative rebinding of dCas9-RNP to target site during dissociation. dCas9-RNA_C (20 nM) was injected over the surface, and dissociation was monitored in the absence (dark orange) or presence (blue) of excess non-biotinylated duplex containing target site C (competitor). As control, dCas9-RNA_C was mixed with excess competitor before being injected over the surface (light orange). (F, G) Time courses for hemimethylated GATC site incision on 0.5 nM GT#2 ( F ) and 0.5 nM GT#2b ( G ) by 50 nM MutS, 50 nM MutL and 25 nM MutH in the absence (left) and presence (right) of a dCas9 roadblock placed on target site C in between the GATC sites. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs underneath the gels show fractions of product containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments.
    Scai Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 2 article reviews
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    Effect of dCas9 roadblock on incision of GATC sites during initiation of DNA mismatch repair. ( A ) DNA substrates contain a single G/T mismatch and Alexa 647 fluorophore at different positions flanked by two hemi-methylated GATC sites and are linearized using ScaI. ( B ) Time courses for strand incision by 50 nM MutS, 50 nM MutL and 25 nM MutH on 0.5 nM GT#2 (left panel; substrate carrying mismatch) or AT#2 (right panel; control containing the corresponding A/T Watson-Crick base pair). Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using Alexa 647 fluorophore emission. ( C ) Schematic of the binding and disassociation of dCas9 RNP complexes on target DNA during surface plasmon resonance analysis using Biacore T100. ( D ) Left panel: dCas9-RNA_A complex (0.6–20 nM) injected over 60-bp duplex DNA carrying target site C. Right panel: dCas9-RNA_C complex (0.6–20 nM) injected over 60-bp duplex DNA carrying site (C). ( E ) Control addressing putative rebinding of dCas9-RNP to target site during dissociation. dCas9-RNA_C (20 nM) was injected over the surface, and dissociation was monitored in the absence (dark orange) or presence (blue) of excess non-biotinylated duplex containing target site C (competitor). As control, dCas9-RNA_C was mixed with excess competitor before being injected over the surface (light orange). (F, G) Time courses for hemimethylated GATC site incision on 0.5 nM GT#2 ( F ) and 0.5 nM GT#2b ( G ) by 50 nM MutS, 50 nM MutL and 25 nM MutH in the absence (left) and presence (right) of a dCas9 roadblock placed on target site C in between the GATC sites. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs underneath the gels show fractions of product containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

    doi: 10.1093/nar/gkz834

    Figure Lengend Snippet: Effect of dCas9 roadblock on incision of GATC sites during initiation of DNA mismatch repair. ( A ) DNA substrates contain a single G/T mismatch and Alexa 647 fluorophore at different positions flanked by two hemi-methylated GATC sites and are linearized using ScaI. ( B ) Time courses for strand incision by 50 nM MutS, 50 nM MutL and 25 nM MutH on 0.5 nM GT#2 (left panel; substrate carrying mismatch) or AT#2 (right panel; control containing the corresponding A/T Watson-Crick base pair). Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using Alexa 647 fluorophore emission. ( C ) Schematic of the binding and disassociation of dCas9 RNP complexes on target DNA during surface plasmon resonance analysis using Biacore T100. ( D ) Left panel: dCas9-RNA_A complex (0.6–20 nM) injected over 60-bp duplex DNA carrying target site C. Right panel: dCas9-RNA_C complex (0.6–20 nM) injected over 60-bp duplex DNA carrying site (C). ( E ) Control addressing putative rebinding of dCas9-RNP to target site during dissociation. dCas9-RNA_C (20 nM) was injected over the surface, and dissociation was monitored in the absence (dark orange) or presence (blue) of excess non-biotinylated duplex containing target site C (competitor). As control, dCas9-RNA_C was mixed with excess competitor before being injected over the surface (light orange). (F, G) Time courses for hemimethylated GATC site incision on 0.5 nM GT#2 ( F ) and 0.5 nM GT#2b ( G ) by 50 nM MutS, 50 nM MutL and 25 nM MutH in the absence (left) and presence (right) of a dCas9 roadblock placed on target site C in between the GATC sites. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs underneath the gels show fractions of product containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments.

    Article Snippet: The fragment indicative for nicking at GATC site 2 was created by digesting GT#2647 using ScaI-HF and BamHI for 30 min at at 37°C.

    Techniques: Methylation, Nucleic Acid Electrophoresis, Binding Assay, SPR Assay, Injection

    In vivo recombination of plasmids by mutants of Cre a) Plasmid architecture of the three expected recombination products. Recombination sites are shown as triangles. For simplicity, the map is in linear form, where the NcoI sites at the ends represent a single NcoI site on the circular plasmids. The numbers under each segment indicate distance in bp (map not drawn to scale). The numbers adjacent to each map are the sizes of the expected digestion products, with the asterisks indicating the product size that is unique to the particular configuration. b) Digest analysis of loxP x loxP (left five lanes) and ψlox h7q21 x ψCore h7q21 (right five lanes) recombination. (c d) Inversion and recombination frequency of (c) loxP x loxP and (d) ψCore h7q21 x ψlox h7q21 recombination obtained by quantifying band intensities. Error bars correspond to 95% C.I. (n= 2 independent experiment). NI: NcoI site; SI: ScaI site.

    Journal: Nature communications

    Article Title: Mutants of Cre recombinase with improved accuracy

    doi: 10.1038/ncomms3509

    Figure Lengend Snippet: In vivo recombination of plasmids by mutants of Cre a) Plasmid architecture of the three expected recombination products. Recombination sites are shown as triangles. For simplicity, the map is in linear form, where the NcoI sites at the ends represent a single NcoI site on the circular plasmids. The numbers under each segment indicate distance in bp (map not drawn to scale). The numbers adjacent to each map are the sizes of the expected digestion products, with the asterisks indicating the product size that is unique to the particular configuration. b) Digest analysis of loxP x loxP (left five lanes) and ψlox h7q21 x ψCore h7q21 (right five lanes) recombination. (c d) Inversion and recombination frequency of (c) loxP x loxP and (d) ψCore h7q21 x ψlox h7q21 recombination obtained by quantifying band intensities. Error bars correspond to 95% C.I. (n= 2 independent experiment). NI: NcoI site; SI: ScaI site.

    Article Snippet: The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9).

    Techniques: In Vivo, Plasmid Preparation

    lgc-35 (L324S) CRISPR-Cas9-RNP genotyping and Mendelian segregation. (A) Top, exon-intron structure of the lgc-35 genomic locus and the PCR strategy used for identifying edited animals. PCR primers were designed to produce a single 820 bp product that flanks the edit of interest. Wild-type alleles (+/+) lacking the edited and ScaI restriction site show a single 820 bp band. Successfully edited heterozygotes (m/+) produce three bands (820, 512, and 308 bp), and homozygotes (m/m) produce two bands (512 and 308 bp) when digested with ScaI. Bottom, genotyping data from the 24 positive red fluorescent progeny chosen from three different P0 injected animals (D, L, I). PCR restriction digest analyses revealed that F1 animals D-2,4,5,7,8, L-2, and I-3 are heterozygous knock-ins. An N2 control is shown at the far right of the gel. PCR failures occurred for animals D-3, L-1,8, and I-6. (B) Representative chromatogram from heterozygous animal D-2. Nucleotide codon spacing and amino acid translations are shown above for the heterozygote and wild-type strands. The desired edit is highlighted by a red box, and the conservative changes that create an in-frame ScaI restriction site are highlighted by a yellow box. (C) Left, Mendelian analysis from animal D-2. 96 single F2 progeny were picked and genotyped. The total for the analysis was 81, since there were 15 PCR failures. Mendelian segregation reveals 17 wild-type (+/+), 46 heterozygote (m/+), and 18 homozygote (m/m) animals. The green outline box shows representative wild type (+/+), the blue outline box shows representative heterozygotes (m/+), and red outline box shows representative homozygotes (m/m). Right, representative chromatogram from a homozygous knock-in animal.

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans

    doi: 10.1534/g3.117.300216

    Figure Lengend Snippet: lgc-35 (L324S) CRISPR-Cas9-RNP genotyping and Mendelian segregation. (A) Top, exon-intron structure of the lgc-35 genomic locus and the PCR strategy used for identifying edited animals. PCR primers were designed to produce a single 820 bp product that flanks the edit of interest. Wild-type alleles (+/+) lacking the edited and ScaI restriction site show a single 820 bp band. Successfully edited heterozygotes (m/+) produce three bands (820, 512, and 308 bp), and homozygotes (m/m) produce two bands (512 and 308 bp) when digested with ScaI. Bottom, genotyping data from the 24 positive red fluorescent progeny chosen from three different P0 injected animals (D, L, I). PCR restriction digest analyses revealed that F1 animals D-2,4,5,7,8, L-2, and I-3 are heterozygous knock-ins. An N2 control is shown at the far right of the gel. PCR failures occurred for animals D-3, L-1,8, and I-6. (B) Representative chromatogram from heterozygous animal D-2. Nucleotide codon spacing and amino acid translations are shown above for the heterozygote and wild-type strands. The desired edit is highlighted by a red box, and the conservative changes that create an in-frame ScaI restriction site are highlighted by a yellow box. (C) Left, Mendelian analysis from animal D-2. 96 single F2 progeny were picked and genotyped. The total for the analysis was 81, since there were 15 PCR failures. Mendelian segregation reveals 17 wild-type (+/+), 46 heterozygote (m/+), and 18 homozygote (m/m) animals. The green outline box shows representative wild type (+/+), the blue outline box shows representative heterozygotes (m/+), and red outline box shows representative homozygotes (m/m). Right, representative chromatogram from a homozygous knock-in animal.

    Article Snippet: The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel.

    Techniques: CRISPR, Polymerase Chain Reaction, Injection, Knock-In