msci  (New England Biolabs)


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    Name:
    MscI
    Description:
    MscI 1 250 units
    Catalog Number:
    R0534L
    Price:
    290
    Category:
    Restriction Enzymes
    Size:
    1 250 units
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    New England Biolabs msci
    MscI
    MscI 1 250 units
    https://www.bioz.com/result/msci/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    msci - by Bioz Stars, 2021-05
    98/100 stars

    Images

    1) Product Images from "Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation"

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation

    Journal: PeerJ

    doi: 10.7717/peerj.224

    Methylation analysis of Moloney vector 5′ LTR using MSRE-qPCR. (A) Sequence of 5′ long terminal repeat (LTR) and untranslated region of Moloney murine leukemia virus. The U3, R and U5 sequences within the LTR are shown and demarcated by vertical lines. Also shown are direct repeats (DR1 and DR2), Tata box, polyadenylation signal (Poly A), negative control region (NCR), binding site for ELP/NR5A1, and primer binding site (PBS). The CpG nucleotides are marked underneath by ‘*’ to indicate putative sites of methylation. The methylation sensitive SmaI and methylation insensitive MscI restriction enzyme sites are shown in red and green, respectively. > > > and
    Figure Legend Snippet: Methylation analysis of Moloney vector 5′ LTR using MSRE-qPCR. (A) Sequence of 5′ long terminal repeat (LTR) and untranslated region of Moloney murine leukemia virus. The U3, R and U5 sequences within the LTR are shown and demarcated by vertical lines. Also shown are direct repeats (DR1 and DR2), Tata box, polyadenylation signal (Poly A), negative control region (NCR), binding site for ELP/NR5A1, and primer binding site (PBS). The CpG nucleotides are marked underneath by ‘*’ to indicate putative sites of methylation. The methylation sensitive SmaI and methylation insensitive MscI restriction enzyme sites are shown in red and green, respectively. > > > and

    Techniques Used: Methylation, Plasmid Preparation, Real-time Polymerase Chain Reaction, Sequencing, Negative Control, Binding Assay

    2) Product Images from "Interethnic diversity of the CD209 (rs4804803) gene promoter polymorphism in African but not American sickle cell disease"

    Article Title: Interethnic diversity of the CD209 (rs4804803) gene promoter polymorphism in African but not American sickle cell disease

    Journal: PeerJ

    doi: 10.7717/peerj.799

    Genotypic distribution of CD209 gene promoter polymorphism (SNP-336 A/G; rs4804803) in Caucasian, African American and African healthy controls. Amplified PCR products were digested with MscI restriction endonuclease (Fisher Scientific, Waltham, Massachusetts, USA), and expressed on a 2% ethidium bromide-stained agarose gel. Homozygote wild type variant (snp-336A) showed no digestion (150 bp); homozygote mutant variant (snp-336G) produced two bands (131 and 19 bp) on digestion (lower band size not shown). Marker: 100 bp ladder, where the 500 bp band stains most intensely (New England Biolabs, Ipswich, Massachusetts, USA). Black bars: Africans; blue bars: African Americans; red bars: Caucasians.
    Figure Legend Snippet: Genotypic distribution of CD209 gene promoter polymorphism (SNP-336 A/G; rs4804803) in Caucasian, African American and African healthy controls. Amplified PCR products were digested with MscI restriction endonuclease (Fisher Scientific, Waltham, Massachusetts, USA), and expressed on a 2% ethidium bromide-stained agarose gel. Homozygote wild type variant (snp-336A) showed no digestion (150 bp); homozygote mutant variant (snp-336G) produced two bands (131 and 19 bp) on digestion (lower band size not shown). Marker: 100 bp ladder, where the 500 bp band stains most intensely (New England Biolabs, Ipswich, Massachusetts, USA). Black bars: Africans; blue bars: African Americans; red bars: Caucasians.

    Techniques Used: Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Variant Assay, Mutagenesis, Produced, Marker

    3) Product Images from "Robust HIV-1 replication in the absence of integrase function"

    Article Title: Robust HIV-1 replication in the absence of integrase function

    Journal: bioRxiv

    doi: 10.1101/2020.03.18.997023

    Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.
    Figure Legend Snippet: Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.

    Techniques Used: Southern Blot, Binding Assay, Infection, Plasmid Preparation, Nucleic Acid Electrophoresis

    HTLV-1 Tax does not facilitate the illegitimate integration of IN- HIV-1. A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN- HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. C) Total 2LTR circle DNA was measured at the indicated timepoints by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN- HIV-1 in the presence or absence of Tax. D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells ± Tax infected with WT or IN- HIV-1. All data in A, C and D are normalized to WT infected Tax-cells at day 1, set to 1; n=3. Crosses indicate last day viable cells were detected. E) DNA from CEM-SS cells ± Tax expression, infected with WT or IN- HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR + linear unintegrated DNA (2.6-2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains ¼ of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. F ) Quantification of the bands (2LTR+1LTR+linear and 2LTR) in E expressed as a percentage of total HIV-1 DNA, which was set at 100%.
    Figure Legend Snippet: HTLV-1 Tax does not facilitate the illegitimate integration of IN- HIV-1. A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN- HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. C) Total 2LTR circle DNA was measured at the indicated timepoints by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN- HIV-1 in the presence or absence of Tax. D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells ± Tax infected with WT or IN- HIV-1. All data in A, C and D are normalized to WT infected Tax-cells at day 1, set to 1; n=3. Crosses indicate last day viable cells were detected. E) DNA from CEM-SS cells ± Tax expression, infected with WT or IN- HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR + linear unintegrated DNA (2.6-2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains ¼ of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. F ) Quantification of the bands (2LTR+1LTR+linear and 2LTR) in E expressed as a percentage of total HIV-1 DNA, which was set at 100%.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Expressing, Southern Blot

    4) Product Images from "Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity"

    Article Title: Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.03.028

    Characterization of shAAV Genomes and In Vivo Evaluation of shAAV Vectors (A) Schematic of pCis constructs used for AAV production. The mTR was removed from vector constructs to assess the ability of shDNA sequences to create double-stranded shAAV vectors. (B) The predicted sizes of packaged genomes were calculated from the base pair lengths between shDNA sequences and wtTR. (C) Viral genome DNA from purified vectors (∼1.0 × 10 10 GCs) in native (left panel) and alkaline (right panel) agarose gels. (D) EGFP expression in livers of adult mice 3 weeks after intravenous injection of rAAV (1.6 × 10 13 GCs/kg). (E) Southern blot analysis of EcoRI- or MscI-digested liver DNA using an EGFP probe. The MscI site is denoted in (A). Small black arrows, linear rAAV genomes; purple arrows, circular rAAVs; magenta arrows, linearized circular rAAVs; white arrowheads, digested linear rAAVs.
    Figure Legend Snippet: Characterization of shAAV Genomes and In Vivo Evaluation of shAAV Vectors (A) Schematic of pCis constructs used for AAV production. The mTR was removed from vector constructs to assess the ability of shDNA sequences to create double-stranded shAAV vectors. (B) The predicted sizes of packaged genomes were calculated from the base pair lengths between shDNA sequences and wtTR. (C) Viral genome DNA from purified vectors (∼1.0 × 10 10 GCs) in native (left panel) and alkaline (right panel) agarose gels. (D) EGFP expression in livers of adult mice 3 weeks after intravenous injection of rAAV (1.6 × 10 13 GCs/kg). (E) Southern blot analysis of EcoRI- or MscI-digested liver DNA using an EGFP probe. The MscI site is denoted in (A). Small black arrows, linear rAAV genomes; purple arrows, circular rAAVs; magenta arrows, linearized circular rAAVs; white arrowheads, digested linear rAAVs.

    Techniques Used: In Vivo, Construct, Plasmid Preparation, Purification, Expressing, Mouse Assay, Injection, Southern Blot

    5) Product Images from "Reversal of Epigenetic Silencing Allows Robust HIV-1 Replication in the Absence of Integrase Function"

    Article Title: Reversal of Epigenetic Silencing Allows Robust HIV-1 Replication in the Absence of Integrase Function

    Journal: mBio

    doi: 10.1128/mBio.01038-20

    HTLV-1 Tax does not facilitate the illegitimate integration of IN − HIV-1. (A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN − HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. (B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. (C) Total 2LTR circle DNA was measured at the indicated time points by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN − HIV-1 in the presence or absence of Tax. (D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells with or without Tax expression and infected with WT or IN − HIV-1. All data in panels A, C, and D are normalized to WT-infected Tax-negative cells at day 1, set to 1; n = 3. Crosses indicate the last day viable cells were detected. (E) DNA from CEM-SS cells with or without Tax expression, infected with WT or IN − HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR plus linear unintegrated DNA (2.6 to 2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains one-quarter of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. (F) Quantification of the bands (2LTR+1LTR+linear and 2LTR) shown in panel E expressed as a percentage of total HIV-1 DNA, which was set at 100%.
    Figure Legend Snippet: HTLV-1 Tax does not facilitate the illegitimate integration of IN − HIV-1. (A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN − HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. (B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. (C) Total 2LTR circle DNA was measured at the indicated time points by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN − HIV-1 in the presence or absence of Tax. (D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells with or without Tax expression and infected with WT or IN − HIV-1. All data in panels A, C, and D are normalized to WT-infected Tax-negative cells at day 1, set to 1; n = 3. Crosses indicate the last day viable cells were detected. (E) DNA from CEM-SS cells with or without Tax expression, infected with WT or IN − HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR plus linear unintegrated DNA (2.6 to 2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains one-quarter of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. (F) Quantification of the bands (2LTR+1LTR+linear and 2LTR) shown in panel E expressed as a percentage of total HIV-1 DNA, which was set at 100%.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Expressing, Southern Blot

    6) Product Images from "PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation"

    Article Title: PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation

    Journal: Clinical and Developmental Immunology

    doi: 10.1155/2013/383681

    PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).
    Figure Legend Snippet: PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).

    Techniques Used: Amplification, Silver Staining, Molecular Weight, Marker

    Related Articles

    Polymerase Chain Reaction:

    Article Title: The “Extended Brown” Plumage Color Mutant of Blue-Breasted Quail (Coturnix chinensis) is Associated with a Mutation in the Melanocortin 1-Receptor Gene (MC1R)
    Article Snippet: The reaction mixture (20 µl ) contained 1.0 µl of DNA, 2.0 µl of 10 × buffer, 2.0 µl of dNTP mixture, 1.0 µl of each primer, 12.9 µl of ultrapure water, and 0.1 µl (0.5 U) of AmpliTaq Gold DNA Polymerase (Applied Biosystems). .. The PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. After the purification of the PCR products, RFLP analysis was performed with 25.0 µl of the reaction solution that contained 500 ng of the PCR products, 2.5 µl of 10 × CutSmart Buffer (New England Biolabs, MA, USA), 1.0 µl of Msc I (New England Biolabs Japan, Tokyo, Japan), and an adequate volume of ultrapure water. .. After the incubation, 5.0 µl of 6× loading buffer (Takara) was added and the PCR products were electrophoresed on a 2.0% agarose gel with 1× Tris-acetic acid-EDTA running buffer using an electrophoresis apparatus (ATTO, Tokyo, Japan).

    Article Title: A PCR plus restriction enzyme-based technique for detecting target-enzyme mutations at position Pro-106 in glyphosate-resistant Lolium perenne
    Article Snippet: .. The restriction digestion reaction (10 μl) contained 8.5 μl of PCR products, 1 μl of the appropriate buffer and 0.5 μl (5 units) of Msc I and Sau 96I enzymes (NEB, UK) for Gly-dCAPS-F1/ Gly-dCAPS-R1 and Gly-dCAPS-F2/ Gly-dCAPS-R2 primers, respectively. .. The restriction digestion reaction (10μl) for Gly-dCAPS-F3/ Gly-dCAPS-R3 primers contained 7.75 μl of PCR products, 1 μl of buffer and 1.25 μl (2.5 units) of Nla IV enzyme (NEB, UK).

    Purification:

    Article Title: The “Extended Brown” Plumage Color Mutant of Blue-Breasted Quail (Coturnix chinensis) is Associated with a Mutation in the Melanocortin 1-Receptor Gene (MC1R)
    Article Snippet: The reaction mixture (20 µl ) contained 1.0 µl of DNA, 2.0 µl of 10 × buffer, 2.0 µl of dNTP mixture, 1.0 µl of each primer, 12.9 µl of ultrapure water, and 0.1 µl (0.5 U) of AmpliTaq Gold DNA Polymerase (Applied Biosystems). .. The PCR conditions were as follows: denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. After the purification of the PCR products, RFLP analysis was performed with 25.0 µl of the reaction solution that contained 500 ng of the PCR products, 2.5 µl of 10 × CutSmart Buffer (New England Biolabs, MA, USA), 1.0 µl of Msc I (New England Biolabs Japan, Tokyo, Japan), and an adequate volume of ultrapure water. .. After the incubation, 5.0 µl of 6× loading buffer (Takara) was added and the PCR products were electrophoresed on a 2.0% agarose gel with 1× Tris-acetic acid-EDTA running buffer using an electrophoresis apparatus (ATTO, Tokyo, Japan).

    Article Title: Robust HIV-1 replication in the absence of integrase function
    Article Snippet: Briefly, DNA was extracted from CEM-SS or Tet-inducible HTLV-1 Tax CEM-SS cells that were infected with DNaseI-treated WT or IN- HIV-1 at 2dpi using a DNA Miniprep Plus kit (Zymo Research). .. Purified DNA was then digested with MscI, XhoI, and DpnI (NEB) overnight, recovered by standard ethanol DNA precipitation, then 10 µg of DNA was run on a 1% TBE gel. .. The gel was then soaked in denaturation solution (1.5 M NaCl, 0.5 M NaOH) to separate the double stranded DNA strands, before being washed in neutralization buffer (3 M NaCl, 0.5 M Tris-HCl pH 7.0).

    Agarose Gel Electrophoresis:

    Article Title: Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity
    Article Snippet: Low-molecular-weight DNA was extracted from triple plasmid-transfected HEK293 cells and digested to completion with DpnI before Southern blot hybridization to eliminate signal from unreplicated vector genomes. .. To analyze rAAV genomes in mouse livers, 5 μg total liver DNA was digested with EcoRI or MscI (New England Biolabs) and resolved by agarose gel electrophoresis for Southern blot hybridization. .. Genomes were visualized using a FLA-7000 Imager (FUJIFILM).

    Southern Blot:

    Article Title: Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity
    Article Snippet: Low-molecular-weight DNA was extracted from triple plasmid-transfected HEK293 cells and digested to completion with DpnI before Southern blot hybridization to eliminate signal from unreplicated vector genomes. .. To analyze rAAV genomes in mouse livers, 5 μg total liver DNA was digested with EcoRI or MscI (New England Biolabs) and resolved by agarose gel electrophoresis for Southern blot hybridization. .. Genomes were visualized using a FLA-7000 Imager (FUJIFILM).

    Hybridization:

    Article Title: Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity
    Article Snippet: Low-molecular-weight DNA was extracted from triple plasmid-transfected HEK293 cells and digested to completion with DpnI before Southern blot hybridization to eliminate signal from unreplicated vector genomes. .. To analyze rAAV genomes in mouse livers, 5 μg total liver DNA was digested with EcoRI or MscI (New England Biolabs) and resolved by agarose gel electrophoresis for Southern blot hybridization. .. Genomes were visualized using a FLA-7000 Imager (FUJIFILM).

    Methylation:

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation
    Article Snippet: The qPCR was accomplished using the Power SYBR Green Master Mix (Invitrogen/Life Technologies, Grand Island, NY, USA; Catalog number: 4367659) in a 7900HT PCR System (Applied Biosystems). .. Methylation-sensitive restriction enzyme qPCR (MSRE-qPCR) Genomic DNA (60 ng), isolated using DNeasy Kits, was digested with 5 units of SmaI (methylation sensitive) or MscI (methylation insensitive) or incubated in the absence of restriction enzyme (uncut) in the manufacturer supplied buffer (New England Biolabs, Massachusetts, USA) in a 50 µl reaction volume at 37°C for 4 h. The reaction was terminated by inactivating the enzymes at 80°C for 20 min. An aliquot of the digest (5 µl) containing 6 ng of genomic DNA was then used in qPCR using primers (SK160 and SK161, ) targeting the 5′ Moloney murine leukemia virus LTR and 5′ untranslated region (UTR) ( ). .. The PCR was carried out using SsoAdvanced SYBR Green Supermix (Bio-Rad, Hercules, CA, USA; Catalog no. 1725261) in a Bio-Rad CFX96 thermocycler.

    Real-time Polymerase Chain Reaction:

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation
    Article Snippet: The qPCR was accomplished using the Power SYBR Green Master Mix (Invitrogen/Life Technologies, Grand Island, NY, USA; Catalog number: 4367659) in a 7900HT PCR System (Applied Biosystems). .. Methylation-sensitive restriction enzyme qPCR (MSRE-qPCR) Genomic DNA (60 ng), isolated using DNeasy Kits, was digested with 5 units of SmaI (methylation sensitive) or MscI (methylation insensitive) or incubated in the absence of restriction enzyme (uncut) in the manufacturer supplied buffer (New England Biolabs, Massachusetts, USA) in a 50 µl reaction volume at 37°C for 4 h. The reaction was terminated by inactivating the enzymes at 80°C for 20 min. An aliquot of the digest (5 µl) containing 6 ng of genomic DNA was then used in qPCR using primers (SK160 and SK161, ) targeting the 5′ Moloney murine leukemia virus LTR and 5′ untranslated region (UTR) ( ). .. The PCR was carried out using SsoAdvanced SYBR Green Supermix (Bio-Rad, Hercules, CA, USA; Catalog no. 1725261) in a Bio-Rad CFX96 thermocycler.

    Isolation:

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation
    Article Snippet: The qPCR was accomplished using the Power SYBR Green Master Mix (Invitrogen/Life Technologies, Grand Island, NY, USA; Catalog number: 4367659) in a 7900HT PCR System (Applied Biosystems). .. Methylation-sensitive restriction enzyme qPCR (MSRE-qPCR) Genomic DNA (60 ng), isolated using DNeasy Kits, was digested with 5 units of SmaI (methylation sensitive) or MscI (methylation insensitive) or incubated in the absence of restriction enzyme (uncut) in the manufacturer supplied buffer (New England Biolabs, Massachusetts, USA) in a 50 µl reaction volume at 37°C for 4 h. The reaction was terminated by inactivating the enzymes at 80°C for 20 min. An aliquot of the digest (5 µl) containing 6 ng of genomic DNA was then used in qPCR using primers (SK160 and SK161, ) targeting the 5′ Moloney murine leukemia virus LTR and 5′ untranslated region (UTR) ( ). .. The PCR was carried out using SsoAdvanced SYBR Green Supermix (Bio-Rad, Hercules, CA, USA; Catalog no. 1725261) in a Bio-Rad CFX96 thermocycler.

    Incubation:

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation
    Article Snippet: The qPCR was accomplished using the Power SYBR Green Master Mix (Invitrogen/Life Technologies, Grand Island, NY, USA; Catalog number: 4367659) in a 7900HT PCR System (Applied Biosystems). .. Methylation-sensitive restriction enzyme qPCR (MSRE-qPCR) Genomic DNA (60 ng), isolated using DNeasy Kits, was digested with 5 units of SmaI (methylation sensitive) or MscI (methylation insensitive) or incubated in the absence of restriction enzyme (uncut) in the manufacturer supplied buffer (New England Biolabs, Massachusetts, USA) in a 50 µl reaction volume at 37°C for 4 h. The reaction was terminated by inactivating the enzymes at 80°C for 20 min. An aliquot of the digest (5 µl) containing 6 ng of genomic DNA was then used in qPCR using primers (SK160 and SK161, ) targeting the 5′ Moloney murine leukemia virus LTR and 5′ untranslated region (UTR) ( ). .. The PCR was carried out using SsoAdvanced SYBR Green Supermix (Bio-Rad, Hercules, CA, USA; Catalog no. 1725261) in a Bio-Rad CFX96 thermocycler.

    Polymorphism Assay:

    Article Title: Interethnic diversity of the CD209 (rs4804803) gene promoter polymorphism in African but not American sickle cell disease
    Article Snippet: Positive amplification yielded products of 150 bp, with size estimated with a TriDye 100 bp DNA ladder (New England Biolabs, Boston, Massachusetts, USA). .. Restriction fragment length polymorphism assay We utilized the MscI (New England Biolabs, Boston, Massachusetts, USA) restriction endonuclease for restriction fragment length polymorphism analysis of CD209 (DC-SIGN 336A/G) gene promoter variants. .. 10 µl of PCR product was mixed with 0.5 µl of enzyme (5,000 U/ml), 5 µl of 1X CutSmart buffer and incubated at 37 °C for 1 h. Digested products were analyzed on an ethidium bromide-stained agarose gel, and band analysis carried out with a Doc-It LS Image Analysis Software (UVP Life Sciences, Upland, California, USA).

    Clone Assay:

    Article Title: Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes
    Article Snippet: The potential positive clones were confirmed by Southern blot using the same protocol as used in the RFLP analysis, except that the duration of the electrophoresis was 16 h. .. Construction of pCC2-tal clones for sequencing pTALBam HI DNA was partially digested with the restriction enzyme Msc I as follow: 2 μg pTALBam HI plasmid DNA, 5 u of Msc I (New England BioLabs), and 2 μl of digestion buffer were mixed; the final reaction volume was adjusted to 20 μl with water, and digestions were performed at 37 °C for a period of time and stopped by heating at 65 °C for 10 minutes and supplementing with 3 μl of 10% SDS-containing loading dye (Takara). .. The DNA fragments were separated by eletrophoresis in agarose gel and the about 1-kb DNA band was sliced out from the gel and purified using QIAquick Gel Extraction Kit.

    Sequencing:

    Article Title: Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes
    Article Snippet: The potential positive clones were confirmed by Southern blot using the same protocol as used in the RFLP analysis, except that the duration of the electrophoresis was 16 h. .. Construction of pCC2-tal clones for sequencing pTALBam HI DNA was partially digested with the restriction enzyme Msc I as follow: 2 μg pTALBam HI plasmid DNA, 5 u of Msc I (New England BioLabs), and 2 μl of digestion buffer were mixed; the final reaction volume was adjusted to 20 μl with water, and digestions were performed at 37 °C for a period of time and stopped by heating at 65 °C for 10 minutes and supplementing with 3 μl of 10% SDS-containing loading dye (Takara). .. The DNA fragments were separated by eletrophoresis in agarose gel and the about 1-kb DNA band was sliced out from the gel and purified using QIAquick Gel Extraction Kit.

    Plasmid Preparation:

    Article Title: Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes
    Article Snippet: The potential positive clones were confirmed by Southern blot using the same protocol as used in the RFLP analysis, except that the duration of the electrophoresis was 16 h. .. Construction of pCC2-tal clones for sequencing pTALBam HI DNA was partially digested with the restriction enzyme Msc I as follow: 2 μg pTALBam HI plasmid DNA, 5 u of Msc I (New England BioLabs), and 2 μl of digestion buffer were mixed; the final reaction volume was adjusted to 20 μl with water, and digestions were performed at 37 °C for a period of time and stopped by heating at 65 °C for 10 minutes and supplementing with 3 μl of 10% SDS-containing loading dye (Takara). .. The DNA fragments were separated by eletrophoresis in agarose gel and the about 1-kb DNA band was sliced out from the gel and purified using QIAquick Gel Extraction Kit.

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    New England Biolabs msc i
    <t>Msc</t> I partial digestion of talC-Bam HI fragment cloned in pUC19. 2 μg plasmid <t>DNA</t> was digested with 5 u Msc I enzyme. The digestion times are given in minutes above the gel. M, 100 bp DNA ladder makers.
    Msc I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Msc I partial digestion of talC-Bam HI fragment cloned in pUC19. 2 μg plasmid DNA was digested with 5 u Msc I enzyme. The digestion times are given in minutes above the gel. M, 100 bp DNA ladder makers.

    Journal: Scientific Reports

    Article Title: Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes

    doi: 10.1038/srep13162

    Figure Lengend Snippet: Msc I partial digestion of talC-Bam HI fragment cloned in pUC19. 2 μg plasmid DNA was digested with 5 u Msc I enzyme. The digestion times are given in minutes above the gel. M, 100 bp DNA ladder makers.

    Article Snippet: Construction of pCC2-tal clones for sequencing pTALBam HI DNA was partially digested with the restriction enzyme Msc I as follow: 2 μg pTALBam HI plasmid DNA, 5 u of Msc I (New England BioLabs), and 2 μl of digestion buffer were mixed; the final reaction volume was adjusted to 20 μl with water, and digestions were performed at 37 °C for a period of time and stopped by heating at 65 °C for 10 minutes and supplementing with 3 μl of 10% SDS-containing loading dye (Takara).

    Techniques: Clone Assay, Plasmid Preparation

    Methylation analysis of Moloney vector 5′ LTR using MSRE-qPCR. (A) Sequence of 5′ long terminal repeat (LTR) and untranslated region of Moloney murine leukemia virus. The U3, R and U5 sequences within the LTR are shown and demarcated by vertical lines. Also shown are direct repeats (DR1 and DR2), Tata box, polyadenylation signal (Poly A), negative control region (NCR), binding site for ELP/NR5A1, and primer binding site (PBS). The CpG nucleotides are marked underneath by ‘*’ to indicate putative sites of methylation. The methylation sensitive SmaI and methylation insensitive MscI restriction enzyme sites are shown in red and green, respectively. > > > and

    Journal: PeerJ

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation

    doi: 10.7717/peerj.224

    Figure Lengend Snippet: Methylation analysis of Moloney vector 5′ LTR using MSRE-qPCR. (A) Sequence of 5′ long terminal repeat (LTR) and untranslated region of Moloney murine leukemia virus. The U3, R and U5 sequences within the LTR are shown and demarcated by vertical lines. Also shown are direct repeats (DR1 and DR2), Tata box, polyadenylation signal (Poly A), negative control region (NCR), binding site for ELP/NR5A1, and primer binding site (PBS). The CpG nucleotides are marked underneath by ‘*’ to indicate putative sites of methylation. The methylation sensitive SmaI and methylation insensitive MscI restriction enzyme sites are shown in red and green, respectively. > > > and

    Article Snippet: Methylation-sensitive restriction enzyme qPCR (MSRE-qPCR) Genomic DNA (60 ng), isolated using DNeasy Kits, was digested with 5 units of SmaI (methylation sensitive) or MscI (methylation insensitive) or incubated in the absence of restriction enzyme (uncut) in the manufacturer supplied buffer (New England Biolabs, Massachusetts, USA) in a 50 µl reaction volume at 37°C for 4 h. The reaction was terminated by inactivating the enzymes at 80°C for 20 min. An aliquot of the digest (5 µl) containing 6 ng of genomic DNA was then used in qPCR using primers (SK160 and SK161, ) targeting the 5′ Moloney murine leukemia virus LTR and 5′ untranslated region (UTR) ( ).

    Techniques: Methylation, Plasmid Preparation, Real-time Polymerase Chain Reaction, Sequencing, Negative Control, Binding Assay

    Genotypic distribution of CD209 gene promoter polymorphism (SNP-336 A/G; rs4804803) in Caucasian, African American and African healthy controls. Amplified PCR products were digested with MscI restriction endonuclease (Fisher Scientific, Waltham, Massachusetts, USA), and expressed on a 2% ethidium bromide-stained agarose gel. Homozygote wild type variant (snp-336A) showed no digestion (150 bp); homozygote mutant variant (snp-336G) produced two bands (131 and 19 bp) on digestion (lower band size not shown). Marker: 100 bp ladder, where the 500 bp band stains most intensely (New England Biolabs, Ipswich, Massachusetts, USA). Black bars: Africans; blue bars: African Americans; red bars: Caucasians.

    Journal: PeerJ

    Article Title: Interethnic diversity of the CD209 (rs4804803) gene promoter polymorphism in African but not American sickle cell disease

    doi: 10.7717/peerj.799

    Figure Lengend Snippet: Genotypic distribution of CD209 gene promoter polymorphism (SNP-336 A/G; rs4804803) in Caucasian, African American and African healthy controls. Amplified PCR products were digested with MscI restriction endonuclease (Fisher Scientific, Waltham, Massachusetts, USA), and expressed on a 2% ethidium bromide-stained agarose gel. Homozygote wild type variant (snp-336A) showed no digestion (150 bp); homozygote mutant variant (snp-336G) produced two bands (131 and 19 bp) on digestion (lower band size not shown). Marker: 100 bp ladder, where the 500 bp band stains most intensely (New England Biolabs, Ipswich, Massachusetts, USA). Black bars: Africans; blue bars: African Americans; red bars: Caucasians.

    Article Snippet: Restriction fragment length polymorphism assay We utilized the MscI (New England Biolabs, Boston, Massachusetts, USA) restriction endonuclease for restriction fragment length polymorphism analysis of CD209 (DC-SIGN 336A/G) gene promoter variants.

    Techniques: Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Variant Assay, Mutagenesis, Produced, Marker

    Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.

    Journal: bioRxiv

    Article Title: Robust HIV-1 replication in the absence of integrase function

    doi: 10.1101/2020.03.18.997023

    Figure Lengend Snippet: Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.

    Article Snippet: Purified DNA was then digested with MscI, XhoI, and DpnI (NEB) overnight, recovered by standard ethanol DNA precipitation, then 10 µg of DNA was run on a 1% TBE gel.

    Techniques: Southern Blot, Binding Assay, Infection, Plasmid Preparation, Nucleic Acid Electrophoresis

    HTLV-1 Tax does not facilitate the illegitimate integration of IN- HIV-1. A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN- HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. C) Total 2LTR circle DNA was measured at the indicated timepoints by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN- HIV-1 in the presence or absence of Tax. D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells ± Tax infected with WT or IN- HIV-1. All data in A, C and D are normalized to WT infected Tax-cells at day 1, set to 1; n=3. Crosses indicate last day viable cells were detected. E) DNA from CEM-SS cells ± Tax expression, infected with WT or IN- HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR + linear unintegrated DNA (2.6-2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains ¼ of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. F ) Quantification of the bands (2LTR+1LTR+linear and 2LTR) in E expressed as a percentage of total HIV-1 DNA, which was set at 100%.

    Journal: bioRxiv

    Article Title: Robust HIV-1 replication in the absence of integrase function

    doi: 10.1101/2020.03.18.997023

    Figure Lengend Snippet: HTLV-1 Tax does not facilitate the illegitimate integration of IN- HIV-1. A) Quantification of integrated HIV-1 DNA in Tet-inducible CEM-SS Tax cells infected with WT or IN- HIV-1, in the presence or absence of Tax induction, as measured by Alu-LTR qPCR. B) Schematic showing the production of a short 2LTR transcript (red line) and the full-length transcript (green line) from circular 2LTR HIV-1 DNA. C) Total 2LTR circle DNA was measured at the indicated timepoints by qPCR, using primers that span the U5-U3 junction, in CEM-SS Tax cells infected with WT or IN- HIV-1 in the presence or absence of Tax. D) Quantification of the predicted 2LTR RNA transcript in CEM-SS cells ± Tax infected with WT or IN- HIV-1. All data in A, C and D are normalized to WT infected Tax-cells at day 1, set to 1; n=3. Crosses indicate last day viable cells were detected. E) DNA from CEM-SS cells ± Tax expression, infected with WT or IN- HIV-1, was digested with MscI and XhoI and probed on a Southern blot with HIV-1 probes that detect specific DNA species that correspond to 2LTR DNA (3.4 kb), 1LTR + linear unintegrated DNA (2.6-2.8 kb), and total HIV-1 DNA (1.9 kb). Note that lane 2 contains ¼ of the amount of cellular DNA used in lanes 1 and 3. See Fig. S2 for details. F ) Quantification of the bands (2LTR+1LTR+linear and 2LTR) in E expressed as a percentage of total HIV-1 DNA, which was set at 100%.

    Article Snippet: Purified DNA was then digested with MscI, XhoI, and DpnI (NEB) overnight, recovered by standard ethanol DNA precipitation, then 10 µg of DNA was run on a 1% TBE gel.

    Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing, Southern Blot