sau96i  (New England Biolabs)


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    Name:
    Sau96I
    Description:
    Sau96I 1 000 units
    Catalog Number:
    r0165s
    Price:
    64
    Size:
    1 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs sau96i
    Sau96I
    Sau96I 1 000 units
    https://www.bioz.com/result/sau96i/product/New England Biolabs
    Average 96 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    sau96i - by Bioz Stars, 2020-07
    96/100 stars

    Images

    1) Product Images from "Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo"

    Article Title: Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-1104

    The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).
    Figure Legend Snippet: The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Sequencing, Derivative Assay

    2) Product Images from "Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis"

    Article Title: Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20175997

    Nested PCR-RFLP (restriction fragment length polymorphism) of SSU rRNA gene for the identification of E . histolytica and E. dispar . Lane 1 , molecular weight marker; lanes 2–5 , Dra I digested PCR products; lane 6 , DNA positive control for E. histolytica (c+eh); lane 7–10 , Sau 96I digested PCR products; lane 11 , DNA positive control for E. dispar (c+ed); and lane 12 , negative control.
    Figure Legend Snippet: Nested PCR-RFLP (restriction fragment length polymorphism) of SSU rRNA gene for the identification of E . histolytica and E. dispar . Lane 1 , molecular weight marker; lanes 2–5 , Dra I digested PCR products; lane 6 , DNA positive control for E. histolytica (c+eh); lane 7–10 , Sau 96I digested PCR products; lane 11 , DNA positive control for E. dispar (c+ed); and lane 12 , negative control.

    Techniques Used: Nested PCR, Molecular Weight, Marker, Polymerase Chain Reaction, Positive Control, Negative Control

    3) Product Images from "Molecular Typing of Australian Scedosporium Isolates Showing Genetic Variability and Numerous S. aurantiacum"

    Article Title: Molecular Typing of Australian Scedosporium Isolates Showing Genetic Variability and Numerous S. aurantiacum

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid1402.070920

    Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) patterns obtained by double digestion with the enzymes Sau 96I and Hha I (A) and of the PCR fingerprinting profiles obtained with the microsatellite specific primer M13 (B) for Scedosporium prolificans : lane 1, WM 06.457; lane 2, WM 06.458; lane 3, WM 06.503; lane 4, WM 06.502; lane 5, WM 06.399; lane 6, WM 06.434. S. aurantiacum : lane 7, WM 06.495; lane 8, WM 06.496; lane 9, WM 06.386; lane 10, WM 06.385; lane 11, WM 06.482; lane 12, WM 06.390. S. apiospermum : lane 13, WM 06.475; lane 14, WM 06.474; lane 15, WM 06.472; lane 16, WM 06.471; lane 17, WM 06.424; lane 18, WM 06.443; lane M, 1-kb marker (GIBCO-BRL, Gaithersburg, MD, USA).
    Figure Legend Snippet: Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) patterns obtained by double digestion with the enzymes Sau 96I and Hha I (A) and of the PCR fingerprinting profiles obtained with the microsatellite specific primer M13 (B) for Scedosporium prolificans : lane 1, WM 06.457; lane 2, WM 06.458; lane 3, WM 06.503; lane 4, WM 06.502; lane 5, WM 06.399; lane 6, WM 06.434. S. aurantiacum : lane 7, WM 06.495; lane 8, WM 06.496; lane 9, WM 06.386; lane 10, WM 06.385; lane 11, WM 06.482; lane 12, WM 06.390. S. apiospermum : lane 13, WM 06.475; lane 14, WM 06.474; lane 15, WM 06.472; lane 16, WM 06.471; lane 17, WM 06.424; lane 18, WM 06.443; lane M, 1-kb marker (GIBCO-BRL, Gaithersburg, MD, USA).

    Techniques Used: Polymerase Chain Reaction, Marker

    4) Product Images from "Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy"

    Article Title: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0228072

    DNA and RNA analysis revealed HDR-mediated gene editing in GRMD-treated (Tx) myoblasts. (a) Agarose gel after PCR and restriction digest of GRMD-Tx and non-Tx cells. From left to right: Normal (N), carrier (Ca), non-Tx GRMD (md), GRMD-sgRNA A-Tx (A), GRMD-sgRNA B-Tx (B), GRMD-sgRNA A B combined Tx (A B), GRMD-TALEN Tx (T), ladder (100bp). All bands were sequenced: top band of ~ 700bp (red asterisk) matched part of the sequence of the donor clone after being cut with Sau96I enzyme, second band of ~ 500bp (red cross) was the corrected DMD gene sequence in GRMD-HDR-Tx samples and normal dog cells. This band was not cut with Sau96I. This second band had a higher molecular weight in GRMD-Tx cells compared to normal due to additional genes (eGFP) present in the donor clone. Third and fourth bands ~ 200bp (red dash, red cash sign) correspond to fragments of the GRMD mutated dog genome that was cut with Sau96I. (b) Sanger sequencing from the cut band (red cross) in a normal dog. Red arrow denotes the correct bp (A) in the DMD gene. (c) Sanger sequencing from the cut band in GRMD-HDR-Tx myoblasts but not successfully edited. Red arrow points at mutated bp (G) in GRMD dogs (d) Sanger sequencing from cut band in successfully GRMD-HDR-Tx GRMD cells. Red arrow denotes successfully replaced bp (A). (e) Dystrophin mRNA expression (mean±SE) among HDR treatments and normal cells compared to normal cells expression. *** p ≤ 0.001, ** p ≤ 0.01 vs Normal. Samples were analyzed using a pair wise fixed reallocation randomization test, excluding outliers with a Grubb’s test. Vertical bars indicate standard error of the mean. Treated myoblasts were differentiated into myotubes for 18 to 21 days and RNA was extracted from 6 replicates; values were normalized to HPRT1 (house-keeping gene).
    Figure Legend Snippet: DNA and RNA analysis revealed HDR-mediated gene editing in GRMD-treated (Tx) myoblasts. (a) Agarose gel after PCR and restriction digest of GRMD-Tx and non-Tx cells. From left to right: Normal (N), carrier (Ca), non-Tx GRMD (md), GRMD-sgRNA A-Tx (A), GRMD-sgRNA B-Tx (B), GRMD-sgRNA A B combined Tx (A B), GRMD-TALEN Tx (T), ladder (100bp). All bands were sequenced: top band of ~ 700bp (red asterisk) matched part of the sequence of the donor clone after being cut with Sau96I enzyme, second band of ~ 500bp (red cross) was the corrected DMD gene sequence in GRMD-HDR-Tx samples and normal dog cells. This band was not cut with Sau96I. This second band had a higher molecular weight in GRMD-Tx cells compared to normal due to additional genes (eGFP) present in the donor clone. Third and fourth bands ~ 200bp (red dash, red cash sign) correspond to fragments of the GRMD mutated dog genome that was cut with Sau96I. (b) Sanger sequencing from the cut band (red cross) in a normal dog. Red arrow denotes the correct bp (A) in the DMD gene. (c) Sanger sequencing from the cut band in GRMD-HDR-Tx myoblasts but not successfully edited. Red arrow points at mutated bp (G) in GRMD dogs (d) Sanger sequencing from cut band in successfully GRMD-HDR-Tx GRMD cells. Red arrow denotes successfully replaced bp (A). (e) Dystrophin mRNA expression (mean±SE) among HDR treatments and normal cells compared to normal cells expression. *** p ≤ 0.001, ** p ≤ 0.01 vs Normal. Samples were analyzed using a pair wise fixed reallocation randomization test, excluding outliers with a Grubb’s test. Vertical bars indicate standard error of the mean. Treated myoblasts were differentiated into myotubes for 18 to 21 days and RNA was extracted from 6 replicates; values were normalized to HPRT1 (house-keeping gene).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, Molecular Weight, Expressing

    Experimental design of HDR-mediated CRISPR/Cas9 and TALEN gene editing for the GRMD mutation. (a) Guide selection included sgRNA A (PAM A underlined) and sgRNA B (PAM B underlined). (b) Experimental design. Double stranded breaks (DSB) occurred at the intron 6 area (highlighted) and/or at the exon 7 area to excise the GRMD mutation (asterisk). The donor clone (green) was used as a template for HDR to replace the excised area with the corrected DMD gene sequence. The black arrow designates the cutting site for Sau96I restriction enzyme, used to genotype GRMD dogs. When the dog does not have the mutated bp, Sau96I does not cut the DNA. (c) TALEN arm design with left and right sequences.
    Figure Legend Snippet: Experimental design of HDR-mediated CRISPR/Cas9 and TALEN gene editing for the GRMD mutation. (a) Guide selection included sgRNA A (PAM A underlined) and sgRNA B (PAM B underlined). (b) Experimental design. Double stranded breaks (DSB) occurred at the intron 6 area (highlighted) and/or at the exon 7 area to excise the GRMD mutation (asterisk). The donor clone (green) was used as a template for HDR to replace the excised area with the corrected DMD gene sequence. The black arrow designates the cutting site for Sau96I restriction enzyme, used to genotype GRMD dogs. When the dog does not have the mutated bp, Sau96I does not cut the DNA. (c) TALEN arm design with left and right sequences.

    Techniques Used: CRISPR, Mutagenesis, Selection, Sequencing

    5) Product Images from "Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo"

    Article Title: Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-1104

    The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).
    Figure Legend Snippet: The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Sequencing, Derivative Assay

    6) Product Images from "Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences"

    Article Title: Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.6.2248-2253.2001

    Genotyping E. cuniculi isolates by PCR analysis of the PTP gene. (a) Differentiation of genotype III from genotypes I and II by eletrophoresis of PCR products: lanes 1 and 10, 100-bp ladders; lanes 2 (strain I), 4 (CDC:V385), 6 (CDC:V446), and 9 (CDC:V428A), genotype I; lane 8 (strain II), genotype II; and lanes 3 (CDC:V282), 5 (USP A-1), and 7 (3275), genotype III. (b) Differentiation of genotype I from genotype II by restriction digestion of PCR products with Sau 96I: lanes 2 (strain I), 3 (CDC:V385), 4 (CDC:V446), and 6 (CDC:V428A), genotype I; and lane 5 (strain II), genotype II.
    Figure Legend Snippet: Genotyping E. cuniculi isolates by PCR analysis of the PTP gene. (a) Differentiation of genotype III from genotypes I and II by eletrophoresis of PCR products: lanes 1 and 10, 100-bp ladders; lanes 2 (strain I), 4 (CDC:V385), 6 (CDC:V446), and 9 (CDC:V428A), genotype I; lane 8 (strain II), genotype II; and lanes 3 (CDC:V282), 5 (USP A-1), and 7 (3275), genotype III. (b) Differentiation of genotype I from genotype II by restriction digestion of PCR products with Sau 96I: lanes 2 (strain I), 3 (CDC:V385), 4 (CDC:V446), and 6 (CDC:V428A), genotype I; and lane 5 (strain II), genotype II.

    Techniques Used: Polymerase Chain Reaction

    7) Product Images from "The cyclin B1 gene is actively transcribed during mitosis in HeLa cells"

    Article Title: The cyclin B1 gene is actively transcribed during mitosis in HeLa cells

    Journal: EMBO Reports

    doi: 10.1093/embo-reports/kve223

    Fig. 2. The cyclin B1 promoter is accessible to restriction endonucleases in vivo . Nuclei isolated from asynchronous (Async.) (20 µg of DNA) and permeabilized MSO cells were partially digested with 100 U of Bsa AI ( A ), 100 U of Sau 96I ( B ), 200 U of Xba I ( C ) or 100 U of Sal I ( D ). Purified DNA was fully digested with Nco I and Xba I when Bsa AI was used for the partial digestion, and with Nco I when Xba I was used for the partial digestion. The probe used for the hybridization is shown at the bottom of each panel. Arrows mark the digested and undigested bands. The schematic diagram at the bottom of each panel indicates the position of the digested bands in the cyclin B1 promoter. The percent digestion is shown below each lane.
    Figure Legend Snippet: Fig. 2. The cyclin B1 promoter is accessible to restriction endonucleases in vivo . Nuclei isolated from asynchronous (Async.) (20 µg of DNA) and permeabilized MSO cells were partially digested with 100 U of Bsa AI ( A ), 100 U of Sau 96I ( B ), 200 U of Xba I ( C ) or 100 U of Sal I ( D ). Purified DNA was fully digested with Nco I and Xba I when Bsa AI was used for the partial digestion, and with Nco I when Xba I was used for the partial digestion. The probe used for the hybridization is shown at the bottom of each panel. Arrows mark the digested and undigested bands. The schematic diagram at the bottom of each panel indicates the position of the digested bands in the cyclin B1 promoter. The percent digestion is shown below each lane.

    Techniques Used: In Vivo, Isolation, Purification, Hybridization

    8) Product Images from "The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital"

    Article Title: The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital

    Journal: Revista do Instituto de Medicina Tropical de São Paulo

    doi: 10.1590/S1678-9946201759013

    RFLP profile of the URA5 genes from Cryptococcus sp obtained by double-digestion with HhaI and Sau96I. Column 1: Molecular maker (100 bp). Columns 2 to 7: isolated 2, 4, 5, 6, 7 and 8 (VNI), respectively; column 8: isolated 12 (VGI); Columns 9 to 13: isolates 13, 18, 22, 24 and 25 (VNI), respectively.
    Figure Legend Snippet: RFLP profile of the URA5 genes from Cryptococcus sp obtained by double-digestion with HhaI and Sau96I. Column 1: Molecular maker (100 bp). Columns 2 to 7: isolated 2, 4, 5, 6, 7 and 8 (VNI), respectively; column 8: isolated 12 (VGI); Columns 9 to 13: isolates 13, 18, 22, 24 and 25 (VNI), respectively.

    Techniques Used: Isolation

    9) Product Images from "Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences"

    Article Title: Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.6.2248-2253.2001

    Genotyping E. cuniculi isolates by PCR analysis of the PTP gene. (a) Differentiation of genotype III from genotypes I and II by eletrophoresis of PCR products: lanes 1 and 10, 100-bp ladders; lanes 2 (strain I), 4 (CDC:V385), 6 (CDC:V446), and 9 (CDC:V428A), genotype I; lane 8 (strain II), genotype II; and lanes 3 (CDC:V282), 5 (USP A-1), and 7 (3275), genotype III. (b) Differentiation of genotype I from genotype II by restriction digestion of PCR products with Sau 96I: lanes 2 (strain I), 3 (CDC:V385), 4 (CDC:V446), and 6 (CDC:V428A), genotype I; and lane 5 (strain II), genotype II.
    Figure Legend Snippet: Genotyping E. cuniculi isolates by PCR analysis of the PTP gene. (a) Differentiation of genotype III from genotypes I and II by eletrophoresis of PCR products: lanes 1 and 10, 100-bp ladders; lanes 2 (strain I), 4 (CDC:V385), 6 (CDC:V446), and 9 (CDC:V428A), genotype I; lane 8 (strain II), genotype II; and lanes 3 (CDC:V282), 5 (USP A-1), and 7 (3275), genotype III. (b) Differentiation of genotype I from genotype II by restriction digestion of PCR products with Sau 96I: lanes 2 (strain I), 3 (CDC:V385), 4 (CDC:V446), and 6 (CDC:V428A), genotype I; and lane 5 (strain II), genotype II.

    Techniques Used: Polymerase Chain Reaction

    10) Product Images from "Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences"

    Article Title: Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.6.2248-2253.2001

    Genotyping E. cuniculi isolates by PCR analysis of the PTP gene. (a) Differentiation of genotype III from genotypes I and II by eletrophoresis of PCR products: lanes 1 and 10, 100-bp ladders; lanes 2 (strain I), 4 (CDC:V385), 6 (CDC:V446), and 9 (CDC:V428A), genotype I; lane 8 (strain II), genotype II; and lanes 3 (CDC:V282), 5 (USP A-1), and 7 (3275), genotype III. (b) Differentiation of genotype I from genotype II by restriction digestion of PCR products with Sau 96I: lanes 2 (strain I), 3 (CDC:V385), 4 (CDC:V446), and 6 (CDC:V428A), genotype I; and lane 5 (strain II), genotype II.
    Figure Legend Snippet: Genotyping E. cuniculi isolates by PCR analysis of the PTP gene. (a) Differentiation of genotype III from genotypes I and II by eletrophoresis of PCR products: lanes 1 and 10, 100-bp ladders; lanes 2 (strain I), 4 (CDC:V385), 6 (CDC:V446), and 9 (CDC:V428A), genotype I; lane 8 (strain II), genotype II; and lanes 3 (CDC:V282), 5 (USP A-1), and 7 (3275), genotype III. (b) Differentiation of genotype I from genotype II by restriction digestion of PCR products with Sau 96I: lanes 2 (strain I), 3 (CDC:V385), 4 (CDC:V446), and 6 (CDC:V428A), genotype I; and lane 5 (strain II), genotype II.

    Techniques Used: Polymerase Chain Reaction

    11) Product Images from "Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo"

    Article Title: Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-1104

    The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).
    Figure Legend Snippet: The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Sequencing, Derivative Assay

    12) Product Images from "Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy"

    Article Title: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0228072

    DNA and RNA analysis revealed HDR-mediated gene editing in GRMD-treated (Tx) myoblasts. (a) Agarose gel after PCR and restriction digest of GRMD-Tx and non-Tx cells. From left to right: Normal (N), carrier (Ca), non-Tx GRMD (md), GRMD-sgRNA A-Tx (A), GRMD-sgRNA B-Tx (B), GRMD-sgRNA A B combined Tx (A B), GRMD-TALEN Tx (T), ladder (100bp). All bands were sequenced: top band of ~ 700bp (red asterisk) matched part of the sequence of the donor clone after being cut with Sau96I enzyme, second band of ~ 500bp (red cross) was the corrected DMD gene sequence in GRMD-HDR-Tx samples and normal dog cells. This band was not cut with Sau96I. This second band had a higher molecular weight in GRMD-Tx cells compared to normal due to additional genes (eGFP) present in the donor clone. Third and fourth bands ~ 200bp (red dash, red cash sign) correspond to fragments of the GRMD mutated dog genome that was cut with Sau96I. (b) Sanger sequencing from the cut band (red cross) in a normal dog. Red arrow denotes the correct bp (A) in the DMD gene. (c) Sanger sequencing from the cut band in GRMD-HDR-Tx myoblasts but not successfully edited. Red arrow points at mutated bp (G) in GRMD dogs (d) Sanger sequencing from cut band in successfully GRMD-HDR-Tx GRMD cells. Red arrow denotes successfully replaced bp (A). (e) Dystrophin mRNA expression (mean±SE) among HDR treatments and normal cells compared to normal cells expression. *** p ≤ 0.001, ** p ≤ 0.01 vs Normal. Samples were analyzed using a pair wise fixed reallocation randomization test, excluding outliers with a Grubb’s test. Vertical bars indicate standard error of the mean. Treated myoblasts were differentiated into myotubes for 18 to 21 days and RNA was extracted from 6 replicates; values were normalized to HPRT1 (house-keeping gene).
    Figure Legend Snippet: DNA and RNA analysis revealed HDR-mediated gene editing in GRMD-treated (Tx) myoblasts. (a) Agarose gel after PCR and restriction digest of GRMD-Tx and non-Tx cells. From left to right: Normal (N), carrier (Ca), non-Tx GRMD (md), GRMD-sgRNA A-Tx (A), GRMD-sgRNA B-Tx (B), GRMD-sgRNA A B combined Tx (A B), GRMD-TALEN Tx (T), ladder (100bp). All bands were sequenced: top band of ~ 700bp (red asterisk) matched part of the sequence of the donor clone after being cut with Sau96I enzyme, second band of ~ 500bp (red cross) was the corrected DMD gene sequence in GRMD-HDR-Tx samples and normal dog cells. This band was not cut with Sau96I. This second band had a higher molecular weight in GRMD-Tx cells compared to normal due to additional genes (eGFP) present in the donor clone. Third and fourth bands ~ 200bp (red dash, red cash sign) correspond to fragments of the GRMD mutated dog genome that was cut with Sau96I. (b) Sanger sequencing from the cut band (red cross) in a normal dog. Red arrow denotes the correct bp (A) in the DMD gene. (c) Sanger sequencing from the cut band in GRMD-HDR-Tx myoblasts but not successfully edited. Red arrow points at mutated bp (G) in GRMD dogs (d) Sanger sequencing from cut band in successfully GRMD-HDR-Tx GRMD cells. Red arrow denotes successfully replaced bp (A). (e) Dystrophin mRNA expression (mean±SE) among HDR treatments and normal cells compared to normal cells expression. *** p ≤ 0.001, ** p ≤ 0.01 vs Normal. Samples were analyzed using a pair wise fixed reallocation randomization test, excluding outliers with a Grubb’s test. Vertical bars indicate standard error of the mean. Treated myoblasts were differentiated into myotubes for 18 to 21 days and RNA was extracted from 6 replicates; values were normalized to HPRT1 (house-keeping gene).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, Molecular Weight, Expressing

    Experimental design of HDR-mediated CRISPR/Cas9 and TALEN gene editing for the GRMD mutation. (a) Guide selection included sgRNA A (PAM A underlined) and sgRNA B (PAM B underlined). (b) Experimental design. Double stranded breaks (DSB) occurred at the intron 6 area (highlighted) and/or at the exon 7 area to excise the GRMD mutation (asterisk). The donor clone (green) was used as a template for HDR to replace the excised area with the corrected DMD gene sequence. The black arrow designates the cutting site for Sau96I restriction enzyme, used to genotype GRMD dogs. When the dog does not have the mutated bp, Sau96I does not cut the DNA. (c) TALEN arm design with left and right sequences.
    Figure Legend Snippet: Experimental design of HDR-mediated CRISPR/Cas9 and TALEN gene editing for the GRMD mutation. (a) Guide selection included sgRNA A (PAM A underlined) and sgRNA B (PAM B underlined). (b) Experimental design. Double stranded breaks (DSB) occurred at the intron 6 area (highlighted) and/or at the exon 7 area to excise the GRMD mutation (asterisk). The donor clone (green) was used as a template for HDR to replace the excised area with the corrected DMD gene sequence. The black arrow designates the cutting site for Sau96I restriction enzyme, used to genotype GRMD dogs. When the dog does not have the mutated bp, Sau96I does not cut the DNA. (c) TALEN arm design with left and right sequences.

    Techniques Used: CRISPR, Mutagenesis, Selection, Sequencing

    Related Articles

    Sequencing:

    Article Title: Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo
    Article Snippet: .. Surprisingly, we found that the GGTCC sequence was cleaved more efficiently by Sau96I than GGACC, GGGCC, or GGCCC in digestions of chromatin or naked DNA control samples. ..

    Incubation:

    Article Title: The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital
    Article Snippet: .. Thirty microliters of the reaction amplicon were doubly digested with Sau96I (10 U/µL) and HhaI (20 U/µL) (New England Biolabs, Uniscience, SP, Brazil), and incubated at 37 °C for 3 hours. ..

    Article Title: Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo
    Article Snippet: .. Cells were washed, pelleted, and resuspended in swelling buffer (10 mM Tris pH8.0, 85 mM KCl, 0.5% NP-40, 10 mM MgCl2 ) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. (For testing whether two REs might increase coverage, in one experiment 100 units of Sau96I and 50 units of DdeI were used in digestion). .. Digestion was terminated by adding 40 μl of 10% SDS and 20 μl of 0.5 M EDTA and the chromatin was treated with proteinase K (Ambion) overnight at 55°C.

    Amplification:

    Article Title: The epidemiology of cryptococcosis and the characterization of Cryptococcus neoformans isolated in a Brazilian University Hospital
    Article Snippet: .. Thirty microliters of the reaction amplicon were doubly digested with Sau96I (10 U/µL) and HhaI (20 U/µL) (New England Biolabs, Uniscience, SP, Brazil), and incubated at 37 °C for 3 hours. ..

    other:

    Article Title: Genotyping Encephalitozoon cuniculi by Multilocus Analyses of Genes with Repetitive Sequences
    Article Snippet: Sau 96I was chosen a the restriction enzyme for restriction fragment length polymorphism (RFLP) analysis, which would cleave genotype I products at two sites, leading to three fragments of predicted sizes of 209 bp, 76 bp, and 78 bp, and genotype II at three sites, leading to four fragments of predicted sizes of 131, 78, 76, and 78 bp.

    Article Title: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy
    Article Snippet: Ligated samples were independently sent to Eton without being digested by Sau96I in order to calculate HDR efficiency.

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    New England Biolabs sau96i
    The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended <t>Sau96I</t> site (GNCC).
    Sau96i, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).

    Journal: BMC Genomics

    Article Title: Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

    doi: 10.1186/1471-2164-15-1104

    Figure Lengend Snippet: The RED-seq method for genome-wide measurement of RE accessibility. (A) RED-seq workflow. RSs are shown in red, yellow boxes (Step 3) represent RS-proximal adaptors, dark blue boxes (Step 5) represent RS-distal adaptors, orange circles represent biotin, light blue boxes represent paired-end PCR primers, large blue circles (Step 1) represent nucleosomes, and DNA is shown in black. (B) Ethidium bromide stained agarose gel indicating bulk digestion levels of chromatin and naked DNA. (C) An example FASTQ file is shown to illustrate the near-uniform sequencing of the RS-containing end of each fragment in the library, signified by the large enrichment of G at position 5, and a CC dinucleotide at positions 7 and 8, derived from the cleaved and blunt-ended Sau96I site (GNCC).

    Article Snippet: Cells were washed, pelleted, and resuspended in swelling buffer (10 mM Tris pH8.0, 85 mM KCl, 0.5% NP-40, 10 mM MgCl2 ) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. (For testing whether two REs might increase coverage, in one experiment 100 units of Sau96I and 50 units of DdeI were used in digestion).

    Techniques: Genome Wide, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Sequencing, Derivative Assay

    Nested PCR-RFLP (restriction fragment length polymorphism) of SSU rRNA gene for the identification of E . histolytica and E. dispar . Lane 1 , molecular weight marker; lanes 2–5 , Dra I digested PCR products; lane 6 , DNA positive control for E. histolytica (c+eh); lane 7–10 , Sau 96I digested PCR products; lane 11 , DNA positive control for E. dispar (c+ed); and lane 12 , negative control.

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis

    doi: 10.1590/1414-431X20175997

    Figure Lengend Snippet: Nested PCR-RFLP (restriction fragment length polymorphism) of SSU rRNA gene for the identification of E . histolytica and E. dispar . Lane 1 , molecular weight marker; lanes 2–5 , Dra I digested PCR products; lane 6 , DNA positive control for E. histolytica (c+eh); lane 7–10 , Sau 96I digested PCR products; lane 11 , DNA positive control for E. dispar (c+ed); and lane 12 , negative control.

    Article Snippet: These products were digested with the restriction endonuclease Dra I or Sau 96I (5 U/µL; BIOLabs, New England) during 16 h at 37°C according to the manufacturer's instructions.

    Techniques: Nested PCR, Molecular Weight, Marker, Polymerase Chain Reaction, Positive Control, Negative Control

    Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) patterns obtained by double digestion with the enzymes Sau 96I and Hha I (A) and of the PCR fingerprinting profiles obtained with the microsatellite specific primer M13 (B) for Scedosporium prolificans : lane 1, WM 06.457; lane 2, WM 06.458; lane 3, WM 06.503; lane 4, WM 06.502; lane 5, WM 06.399; lane 6, WM 06.434. S. aurantiacum : lane 7, WM 06.495; lane 8, WM 06.496; lane 9, WM 06.386; lane 10, WM 06.385; lane 11, WM 06.482; lane 12, WM 06.390. S. apiospermum : lane 13, WM 06.475; lane 14, WM 06.474; lane 15, WM 06.472; lane 16, WM 06.471; lane 17, WM 06.424; lane 18, WM 06.443; lane M, 1-kb marker (GIBCO-BRL, Gaithersburg, MD, USA).

    Journal: Emerging Infectious Diseases

    Article Title: Molecular Typing of Australian Scedosporium Isolates Showing Genetic Variability and Numerous S. aurantiacum

    doi: 10.3201/eid1402.070920

    Figure Lengend Snippet: Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) patterns obtained by double digestion with the enzymes Sau 96I and Hha I (A) and of the PCR fingerprinting profiles obtained with the microsatellite specific primer M13 (B) for Scedosporium prolificans : lane 1, WM 06.457; lane 2, WM 06.458; lane 3, WM 06.503; lane 4, WM 06.502; lane 5, WM 06.399; lane 6, WM 06.434. S. aurantiacum : lane 7, WM 06.495; lane 8, WM 06.496; lane 9, WM 06.386; lane 10, WM 06.385; lane 11, WM 06.482; lane 12, WM 06.390. S. apiospermum : lane 13, WM 06.475; lane 14, WM 06.474; lane 15, WM 06.472; lane 16, WM 06.471; lane 17, WM 06.424; lane 18, WM 06.443; lane M, 1-kb marker (GIBCO-BRL, Gaithersburg, MD, USA).

    Article Snippet: Amplicons were double digested with the restriction endonucleases Sau 96I and Hha I (New England BioLabs, Ipswich, MA, USA) in accordance with the manufacturer’s recommendations.

    Techniques: Polymerase Chain Reaction, Marker

    DNA and RNA analysis revealed HDR-mediated gene editing in GRMD-treated (Tx) myoblasts. (a) Agarose gel after PCR and restriction digest of GRMD-Tx and non-Tx cells. From left to right: Normal (N), carrier (Ca), non-Tx GRMD (md), GRMD-sgRNA A-Tx (A), GRMD-sgRNA B-Tx (B), GRMD-sgRNA A B combined Tx (A B), GRMD-TALEN Tx (T), ladder (100bp). All bands were sequenced: top band of ~ 700bp (red asterisk) matched part of the sequence of the donor clone after being cut with Sau96I enzyme, second band of ~ 500bp (red cross) was the corrected DMD gene sequence in GRMD-HDR-Tx samples and normal dog cells. This band was not cut with Sau96I. This second band had a higher molecular weight in GRMD-Tx cells compared to normal due to additional genes (eGFP) present in the donor clone. Third and fourth bands ~ 200bp (red dash, red cash sign) correspond to fragments of the GRMD mutated dog genome that was cut with Sau96I. (b) Sanger sequencing from the cut band (red cross) in a normal dog. Red arrow denotes the correct bp (A) in the DMD gene. (c) Sanger sequencing from the cut band in GRMD-HDR-Tx myoblasts but not successfully edited. Red arrow points at mutated bp (G) in GRMD dogs (d) Sanger sequencing from cut band in successfully GRMD-HDR-Tx GRMD cells. Red arrow denotes successfully replaced bp (A). (e) Dystrophin mRNA expression (mean±SE) among HDR treatments and normal cells compared to normal cells expression. *** p ≤ 0.001, ** p ≤ 0.01 vs Normal. Samples were analyzed using a pair wise fixed reallocation randomization test, excluding outliers with a Grubb’s test. Vertical bars indicate standard error of the mean. Treated myoblasts were differentiated into myotubes for 18 to 21 days and RNA was extracted from 6 replicates; values were normalized to HPRT1 (house-keeping gene).

    Journal: PLoS ONE

    Article Title: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

    doi: 10.1371/journal.pone.0228072

    Figure Lengend Snippet: DNA and RNA analysis revealed HDR-mediated gene editing in GRMD-treated (Tx) myoblasts. (a) Agarose gel after PCR and restriction digest of GRMD-Tx and non-Tx cells. From left to right: Normal (N), carrier (Ca), non-Tx GRMD (md), GRMD-sgRNA A-Tx (A), GRMD-sgRNA B-Tx (B), GRMD-sgRNA A B combined Tx (A B), GRMD-TALEN Tx (T), ladder (100bp). All bands were sequenced: top band of ~ 700bp (red asterisk) matched part of the sequence of the donor clone after being cut with Sau96I enzyme, second band of ~ 500bp (red cross) was the corrected DMD gene sequence in GRMD-HDR-Tx samples and normal dog cells. This band was not cut with Sau96I. This second band had a higher molecular weight in GRMD-Tx cells compared to normal due to additional genes (eGFP) present in the donor clone. Third and fourth bands ~ 200bp (red dash, red cash sign) correspond to fragments of the GRMD mutated dog genome that was cut with Sau96I. (b) Sanger sequencing from the cut band (red cross) in a normal dog. Red arrow denotes the correct bp (A) in the DMD gene. (c) Sanger sequencing from the cut band in GRMD-HDR-Tx myoblasts but not successfully edited. Red arrow points at mutated bp (G) in GRMD dogs (d) Sanger sequencing from cut band in successfully GRMD-HDR-Tx GRMD cells. Red arrow denotes successfully replaced bp (A). (e) Dystrophin mRNA expression (mean±SE) among HDR treatments and normal cells compared to normal cells expression. *** p ≤ 0.001, ** p ≤ 0.01 vs Normal. Samples were analyzed using a pair wise fixed reallocation randomization test, excluding outliers with a Grubb’s test. Vertical bars indicate standard error of the mean. Treated myoblasts were differentiated into myotubes for 18 to 21 days and RNA was extracted from 6 replicates; values were normalized to HPRT1 (house-keeping gene).

    Article Snippet: Ligated samples were independently sent to Eton without being digested by Sau96I in order to calculate HDR efficiency.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, Molecular Weight, Expressing

    Experimental design of HDR-mediated CRISPR/Cas9 and TALEN gene editing for the GRMD mutation. (a) Guide selection included sgRNA A (PAM A underlined) and sgRNA B (PAM B underlined). (b) Experimental design. Double stranded breaks (DSB) occurred at the intron 6 area (highlighted) and/or at the exon 7 area to excise the GRMD mutation (asterisk). The donor clone (green) was used as a template for HDR to replace the excised area with the corrected DMD gene sequence. The black arrow designates the cutting site for Sau96I restriction enzyme, used to genotype GRMD dogs. When the dog does not have the mutated bp, Sau96I does not cut the DNA. (c) TALEN arm design with left and right sequences.

    Journal: PLoS ONE

    Article Title: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy

    doi: 10.1371/journal.pone.0228072

    Figure Lengend Snippet: Experimental design of HDR-mediated CRISPR/Cas9 and TALEN gene editing for the GRMD mutation. (a) Guide selection included sgRNA A (PAM A underlined) and sgRNA B (PAM B underlined). (b) Experimental design. Double stranded breaks (DSB) occurred at the intron 6 area (highlighted) and/or at the exon 7 area to excise the GRMD mutation (asterisk). The donor clone (green) was used as a template for HDR to replace the excised area with the corrected DMD gene sequence. The black arrow designates the cutting site for Sau96I restriction enzyme, used to genotype GRMD dogs. When the dog does not have the mutated bp, Sau96I does not cut the DNA. (c) TALEN arm design with left and right sequences.

    Article Snippet: Ligated samples were independently sent to Eton without being digested by Sau96I in order to calculate HDR efficiency.

    Techniques: CRISPR, Mutagenesis, Selection, Sequencing