aatii  (New England Biolabs)


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    Name:
    AatII
    Description:
    AatII 2 500 units
    Catalog Number:
    r0117l
    Price:
    257
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    Structured Review

    New England Biolabs aatii
    AatII
    AatII 2 500 units
    https://www.bioz.com/result/aatii/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    aatii - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "The Role of Neutrophils in Measles Virus–mediated Oncolysis Differs Between B-cell Malignancies and Is Not Always Enhanced by GCSF"

    Article Title: The Role of Neutrophils in Measles Virus–mediated Oncolysis Differs Between B-cell Malignancies and Is Not Always Enhanced by GCSF

    Journal: Molecular Therapy

    doi: 10.1038/mt.2015.149

    Construction and characterization of MV-expressing hGCSF . ( a ) Schematic showing cloning of DNA encoding human cytokine granulocyte colony-stimulating factor (hGCSF) p (+) MV-NSe upstream of M, using AatII and MluI restriction enzymes. ( b ) One step growth
    Figure Legend Snippet: Construction and characterization of MV-expressing hGCSF . ( a ) Schematic showing cloning of DNA encoding human cytokine granulocyte colony-stimulating factor (hGCSF) p (+) MV-NSe upstream of M, using AatII and MluI restriction enzymes. ( b ) One step growth

    Techniques Used: Expressing, Clone Assay

    2) Product Images from "?-Agonists Selectively Modulate Proinflammatory Gene Expression in Skeletal Muscle Cells via Non-Canonical Nuclear Crosstalk Mechanisms"

    Article Title: ?-Agonists Selectively Modulate Proinflammatory Gene Expression in Skeletal Muscle Cells via Non-Canonical Nuclear Crosstalk Mechanisms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090649

    TNF-α/isoproterenol cotreatment induces chromatin remodelling and histone H3 modifications at the IL-6 promoter in C2C12 myotubes. (A) Schematic representation of the localisation of CREB- and NF-κB-responsive elements in the IL-6 promoter. Relative position of the recognition sites for AatII and HincII and primers used in the restriction enzyme accessibility assay via Real Time PCR (CHART-PCR) are indicated. (B) TNF/iso cotreatment promotes accessibility of the IL-6 promoter. C2C12 cells were treated for 2 hours with veh, iso and/or TNF. Chromatin opening of the promoter region was determined by CHART-PCR as detailed in Materials and Methods . Results represent average ± SD of three independent experiments. (C) Effect of TSA, a histone deacetylase inhibitor, on IL-6 transcription. C2C12 cells were treated for 2 hours with combinations of veh, TSA, iso and/or TNF. mRNA levels of IL-6 were determined via RT-qPCR. Results represent average ± SD of three independent experiments. (D) TNF/iso cotreatment enhances phosphorylation of histone H3 at the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Phosphorylation of histone H3 at serine 10 was determined via ChIP. Results represent average ± SD of three independent experiments. (E-H) Effect of TNF and/or iso treatment on the recruitment of NF-κB, CREB, CBP and RNA polymerase II to the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Recruitment of NF-κB p65, CREB, CBP and RNA polymerase II was measured via ChIP. Results represent average ± SD of three independent experiments. (*) Significantly different from veh. (**) Significantly different from TNF. (***) Significantly different from iso.
    Figure Legend Snippet: TNF-α/isoproterenol cotreatment induces chromatin remodelling and histone H3 modifications at the IL-6 promoter in C2C12 myotubes. (A) Schematic representation of the localisation of CREB- and NF-κB-responsive elements in the IL-6 promoter. Relative position of the recognition sites for AatII and HincII and primers used in the restriction enzyme accessibility assay via Real Time PCR (CHART-PCR) are indicated. (B) TNF/iso cotreatment promotes accessibility of the IL-6 promoter. C2C12 cells were treated for 2 hours with veh, iso and/or TNF. Chromatin opening of the promoter region was determined by CHART-PCR as detailed in Materials and Methods . Results represent average ± SD of three independent experiments. (C) Effect of TSA, a histone deacetylase inhibitor, on IL-6 transcription. C2C12 cells were treated for 2 hours with combinations of veh, TSA, iso and/or TNF. mRNA levels of IL-6 were determined via RT-qPCR. Results represent average ± SD of three independent experiments. (D) TNF/iso cotreatment enhances phosphorylation of histone H3 at the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Phosphorylation of histone H3 at serine 10 was determined via ChIP. Results represent average ± SD of three independent experiments. (E-H) Effect of TNF and/or iso treatment on the recruitment of NF-κB, CREB, CBP and RNA polymerase II to the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Recruitment of NF-κB p65, CREB, CBP and RNA polymerase II was measured via ChIP. Results represent average ± SD of three independent experiments. (*) Significantly different from veh. (**) Significantly different from TNF. (***) Significantly different from iso.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Histone Deacetylase Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation

    3) Product Images from "Defining characteristics of Tn5 Transposase non-specific DNA binding"

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl179

    Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.
    Figure Legend Snippet: Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Techniques Used: Plasmid Preparation, In Vitro, Incubation, Labeling, Agarose Gel Electrophoresis

    4) Product Images from "Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency"

    Article Title: Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency

    Journal:

    doi: 10.1128/JVI.79.8.5069-5077.2005

    Southern blots of virion DNA from cells infected with ORF63 deletion mutants. (A) ROka, ROka63-AatII15-3, and ROka63-AatII30-4 DNAs were digested with BclI and AatII and hybridized to an ORF63 probe. (B) ROka, ROka63-AccI, ROka63-SfoI, and ROka63-KpnI
    Figure Legend Snippet: Southern blots of virion DNA from cells infected with ORF63 deletion mutants. (A) ROka, ROka63-AatII15-3, and ROka63-AatII30-4 DNAs were digested with BclI and AatII and hybridized to an ORF63 probe. (B) ROka, ROka63-AccI, ROka63-SfoI, and ROka63-KpnI

    Techniques Used: Infection

    5) Product Images from "One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome"

    Article Title: One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

    Journal:

    doi: 10.1073/pnas.0811011106

    Validation of an intact M. genitalium genome by restriction analysis. ( A ) Diagram of the EagI (red), BssHII (blue), and AatII (green) restriction fragments expected for a complete and proper assembly of the synthetic JCVI-1.1 M. genitalium genome. The
    Figure Legend Snippet: Validation of an intact M. genitalium genome by restriction analysis. ( A ) Diagram of the EagI (red), BssHII (blue), and AatII (green) restriction fragments expected for a complete and proper assembly of the synthetic JCVI-1.1 M. genitalium genome. The

    Techniques Used:

    6) Product Images from "The CMG helicase bypasses DNA-protein cross-links to facilitate their repair"

    Article Title: The CMG helicase bypasses DNA-protein cross-links to facilitate their repair

    Journal: Cell

    doi: 10.1016/j.cell.2018.10.053

    Disappearance of the CMG footprint at DPC Lead is unaffected by proteolysis or p97 inhibition. ( A ). ( B ). ( C ) pDPC Lead or pmeDPC Lead were pre-bound with LacR to prevent one replication fork from reaching the DPC. The plasmids were replicated in mock-depleted or SPRTN-depleted egg extract containing 32 P[α]-dATP and supplemented with buffer or the p97 inhibitor NMS-873 (p97i). At different times, DNA was recovered and digested with AatII and FspI, separated on a denaturing polyacrylamide gel, and visualized by autoradiography. Grey inset: Schematic of nascent leading strand products released by AatII and FspI digestion of pmeDPC Lead or pDPC Lead for description of −1 to +12 products in lanes 7–12 and 19–24. ( D ) pmeDPC 2xLead .
    Figure Legend Snippet: Disappearance of the CMG footprint at DPC Lead is unaffected by proteolysis or p97 inhibition. ( A ). ( B ). ( C ) pDPC Lead or pmeDPC Lead were pre-bound with LacR to prevent one replication fork from reaching the DPC. The plasmids were replicated in mock-depleted or SPRTN-depleted egg extract containing 32 P[α]-dATP and supplemented with buffer or the p97 inhibitor NMS-873 (p97i). At different times, DNA was recovered and digested with AatII and FspI, separated on a denaturing polyacrylamide gel, and visualized by autoradiography. Grey inset: Schematic of nascent leading strand products released by AatII and FspI digestion of pmeDPC Lead or pDPC Lead for description of −1 to +12 products in lanes 7–12 and 19–24. ( D ) pmeDPC 2xLead .

    Techniques Used: Inhibition, Autoradiography

    Related Articles

    Polymerase Chain Reaction:

    Article Title: The Role of Neutrophils in Measles Virus–mediated Oncolysis Differs Between B-cell Malignancies and Is Not Always Enhanced by GCSF
    Article Snippet: Site-directed mutagenesis was used to remove an Aat II site from the middle of the gene using Stratagene Quickchange II kit (Agilent Technologies, UK). .. It was then PCR amplified with the following primers, forward primer MluI+hGCSF: 5′- agtattac acgcgt atggctggacctgccacccagagc-3′ and reverse primer: AatII_hGCSF: 5′-tacagtcg gacgtc attcagggctgggcaaggtggcg-3′, and the PCR product was cloned into the MVNSe backbone using AatII and MluI restriction endonucleases (New England Biolab, UK), replacing GFP, upstream of MV M gene. ..

    Amplification:

    Article Title: The Role of Neutrophils in Measles Virus–mediated Oncolysis Differs Between B-cell Malignancies and Is Not Always Enhanced by GCSF
    Article Snippet: Site-directed mutagenesis was used to remove an Aat II site from the middle of the gene using Stratagene Quickchange II kit (Agilent Technologies, UK). .. It was then PCR amplified with the following primers, forward primer MluI+hGCSF: 5′- agtattac acgcgt atggctggacctgccacccagagc-3′ and reverse primer: AatII_hGCSF: 5′-tacagtcg gacgtc attcagggctgggcaaggtggcg-3′, and the PCR product was cloned into the MVNSe backbone using AatII and MluI restriction endonucleases (New England Biolab, UK), replacing GFP, upstream of MV M gene. ..

    Clone Assay:

    Article Title: The Role of Neutrophils in Measles Virus–mediated Oncolysis Differs Between B-cell Malignancies and Is Not Always Enhanced by GCSF
    Article Snippet: Site-directed mutagenesis was used to remove an Aat II site from the middle of the gene using Stratagene Quickchange II kit (Agilent Technologies, UK). .. It was then PCR amplified with the following primers, forward primer MluI+hGCSF: 5′- agtattac acgcgt atggctggacctgccacccagagc-3′ and reverse primer: AatII_hGCSF: 5′-tacagtcg gacgtc attcagggctgggcaaggtggcg-3′, and the PCR product was cloned into the MVNSe backbone using AatII and MluI restriction endonucleases (New England Biolab, UK), replacing GFP, upstream of MV M gene. ..

    Article Title: Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency
    Article Snippet: Plasmid 63-BclI, which contains ORF63 (VZV nucleotides 106592 to 112215) within a VZV BclI fragment, was also previously described ( ). .. To construct carboxy-terminal mutants of ORF63, plasmid 63-BclI was digested with HpaI and AatII (which cut at VZV nucleotides 110649 and 111366, respectively), and the insert containing ORF63 was cloned into the corresponding sites of plasmid LITMUS 28i (New England Biolabs, Beverly, Mass.). .. The resulting plasmid was cut with SfoI, AccI, or KpnI, which cut at VZV nucleotides 111005, 111088, and 111208, respectively.

    Construct:

    Article Title: Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency
    Article Snippet: Plasmid 63-BclI, which contains ORF63 (VZV nucleotides 106592 to 112215) within a VZV BclI fragment, was also previously described ( ). .. To construct carboxy-terminal mutants of ORF63, plasmid 63-BclI was digested with HpaI and AatII (which cut at VZV nucleotides 110649 and 111366, respectively), and the insert containing ORF63 was cloned into the corresponding sites of plasmid LITMUS 28i (New England Biolabs, Beverly, Mass.). .. The resulting plasmid was cut with SfoI, AccI, or KpnI, which cut at VZV nucleotides 111005, 111088, and 111208, respectively.

    Plasmid Preparation:

    Article Title: Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency
    Article Snippet: Plasmid 63-BclI, which contains ORF63 (VZV nucleotides 106592 to 112215) within a VZV BclI fragment, was also previously described ( ). .. To construct carboxy-terminal mutants of ORF63, plasmid 63-BclI was digested with HpaI and AatII (which cut at VZV nucleotides 110649 and 111366, respectively), and the insert containing ORF63 was cloned into the corresponding sites of plasmid LITMUS 28i (New England Biolabs, Beverly, Mass.). .. The resulting plasmid was cut with SfoI, AccI, or KpnI, which cut at VZV nucleotides 111005, 111088, and 111208, respectively.

    Article Title: Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters
    Article Snippet: DNA was purified after each step using the QiaQuick Nucleotide Cleanup Kit (Qiagen). .. The rrnB P1 fragment (∼190 bp) was produced by AatII (NEB) digestion of the linear plasmid and gel-isolated as described ( ). .. We incubated 10–20 nM RNAP holoenzyme with ∼0.2 nM promoter fragment in binding buffer (40 mM Tris·HCl pH 7.4, 1 mM DTT, 30 mM NaCl) at 37 °C for 10 min. DTT and Fe2+ (final concentrations 5 mM and 1 μM, respectively) were then added simultaneously.

    Incubation:

    Article Title: The CMG helicase bypasses DNA-protein cross-links to facilitate their repair
    Article Snippet: .. To nick radio-labeled nascent leading-strands, 3–4 μl of extracted and ethanol precipitated DNA (see above) at 1–2 ng μl−1 was incubated in 1x buffer 3.1 (New England BioLabs) with 0.45 units μl−1 Nb.BsmI (New England BioLabs) in a 5 μl reaction at 65 °C for 1 h. To digest radio-labeled nascent leading-strand 3–4 μl of extracted and ethanol precipitated DNA a 1–2 ng μl−1 was incubated in 1x cutsmart buffer (New England BioLabs) with 1 unit μl−1 AatII (New England BioLabs) and FspI (New England BioLabs) in a 5 μl reaction at 37 °C for 2 h. To nick rightward leading strands of pLacO32 , 3–4 μl of purified DNA at 1–2 ng μl−1 was incubated in buffer 3.1 with 0.4 units μl−1 Nt.BspQI (New England BioLabs) at 37 °C for 1 h. Digestion reactions were stopped with 0.5 volumes of Sequencing Stop solution (95% formamide, 20 mM EDTA, 0.05 % bromophenol blue, 0.05% xylene cyanol FF). .. Nicked DNA (3.5 to 4 μl samples) was separated on 4 % (for pLacO) or 7% (pDPC) polyacrylamide sequencing gels.

    Purification:

    Article Title: The CMG helicase bypasses DNA-protein cross-links to facilitate their repair
    Article Snippet: .. To nick radio-labeled nascent leading-strands, 3–4 μl of extracted and ethanol precipitated DNA (see above) at 1–2 ng μl−1 was incubated in 1x buffer 3.1 (New England BioLabs) with 0.45 units μl−1 Nb.BsmI (New England BioLabs) in a 5 μl reaction at 65 °C for 1 h. To digest radio-labeled nascent leading-strand 3–4 μl of extracted and ethanol precipitated DNA a 1–2 ng μl−1 was incubated in 1x cutsmart buffer (New England BioLabs) with 1 unit μl−1 AatII (New England BioLabs) and FspI (New England BioLabs) in a 5 μl reaction at 37 °C for 2 h. To nick rightward leading strands of pLacO32 , 3–4 μl of purified DNA at 1–2 ng μl−1 was incubated in buffer 3.1 with 0.4 units μl−1 Nt.BspQI (New England BioLabs) at 37 °C for 1 h. Digestion reactions were stopped with 0.5 volumes of Sequencing Stop solution (95% formamide, 20 mM EDTA, 0.05 % bromophenol blue, 0.05% xylene cyanol FF). .. Nicked DNA (3.5 to 4 μl samples) was separated on 4 % (for pLacO) or 7% (pDPC) polyacrylamide sequencing gels.

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science
    Article Snippet: For each TALEN, one or two PCR-positive colonies were selected and cultured in LB + carbenicillin overnight. .. Day 3: Plasmids were purified from overnight cultures and double-digested with Aat II and Stu I (New England BioLabs) to confirm the presence and size of TALE domains. ..

    Sequencing:

    Article Title: The CMG helicase bypasses DNA-protein cross-links to facilitate their repair
    Article Snippet: .. To nick radio-labeled nascent leading-strands, 3–4 μl of extracted and ethanol precipitated DNA (see above) at 1–2 ng μl−1 was incubated in 1x buffer 3.1 (New England BioLabs) with 0.45 units μl−1 Nb.BsmI (New England BioLabs) in a 5 μl reaction at 65 °C for 1 h. To digest radio-labeled nascent leading-strand 3–4 μl of extracted and ethanol precipitated DNA a 1–2 ng μl−1 was incubated in 1x cutsmart buffer (New England BioLabs) with 1 unit μl−1 AatII (New England BioLabs) and FspI (New England BioLabs) in a 5 μl reaction at 37 °C for 2 h. To nick rightward leading strands of pLacO32 , 3–4 μl of purified DNA at 1–2 ng μl−1 was incubated in buffer 3.1 with 0.4 units μl−1 Nt.BspQI (New England BioLabs) at 37 °C for 1 h. Digestion reactions were stopped with 0.5 volumes of Sequencing Stop solution (95% formamide, 20 mM EDTA, 0.05 % bromophenol blue, 0.05% xylene cyanol FF). .. Nicked DNA (3.5 to 4 μl samples) was separated on 4 % (for pLacO) or 7% (pDPC) polyacrylamide sequencing gels.

    Produced:

    Article Title: Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters
    Article Snippet: DNA was purified after each step using the QiaQuick Nucleotide Cleanup Kit (Qiagen). .. The rrnB P1 fragment (∼190 bp) was produced by AatII (NEB) digestion of the linear plasmid and gel-isolated as described ( ). .. We incubated 10–20 nM RNAP holoenzyme with ∼0.2 nM promoter fragment in binding buffer (40 mM Tris·HCl pH 7.4, 1 mM DTT, 30 mM NaCl) at 37 °C for 10 min. DTT and Fe2+ (final concentrations 5 mM and 1 μM, respectively) were then added simultaneously.

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    New England Biolabs aat ii
    Quality control of TALEN assembly, using colony PCR and restriction enzyme digestion. (A) Representative colony PCR using primers TAL_F1/TAL_R2. Correct colonies (+) result in characteristic laddering due to the repetitive nature of the incorporated RVDs. (B) Representative restriction enzyme digestion analyses with <t>Aat</t> II/ <t>Stu</t> I are shown [these clones differ from those shown in (A) ]. The size of the lower band is dependent on the number of TALE repeats within the TALEN, which can aid in determining correct clones. +, correct TALEN clones; *, incorrect TALEN clones.
    Aat Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aat ii/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    aat ii - by Bioz Stars, 2021-03
    94/100 stars
      Buy from Supplier

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    Quality control of TALEN assembly, using colony PCR and restriction enzyme digestion. (A) Representative colony PCR using primers TAL_F1/TAL_R2. Correct colonies (+) result in characteristic laddering due to the repetitive nature of the incorporated RVDs. (B) Representative restriction enzyme digestion analyses with Aat II/ Stu I are shown [these clones differ from those shown in (A) ]. The size of the lower band is dependent on the number of TALE repeats within the TALEN, which can aid in determining correct clones. +, correct TALEN clones; *, incorrect TALEN clones.

    Journal: Human Gene Therapy

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

    doi: 10.1089/hum.2015.172

    Figure Lengend Snippet: Quality control of TALEN assembly, using colony PCR and restriction enzyme digestion. (A) Representative colony PCR using primers TAL_F1/TAL_R2. Correct colonies (+) result in characteristic laddering due to the repetitive nature of the incorporated RVDs. (B) Representative restriction enzyme digestion analyses with Aat II/ Stu I are shown [these clones differ from those shown in (A) ]. The size of the lower band is dependent on the number of TALE repeats within the TALEN, which can aid in determining correct clones. +, correct TALEN clones; *, incorrect TALEN clones.

    Article Snippet: Day 3: Plasmids were purified from overnight cultures and double-digested with Aat II and Stu I (New England BioLabs) to confirm the presence and size of TALE domains.

    Techniques: Polymerase Chain Reaction, Clone Assay

    Construction and characterization of MV-expressing hGCSF . ( a ) Schematic showing cloning of DNA encoding human cytokine granulocyte colony-stimulating factor (hGCSF) p (+) MV-NSe upstream of M, using AatII and MluI restriction enzymes. ( b ) One step growth

    Journal: Molecular Therapy

    Article Title: The Role of Neutrophils in Measles Virus–mediated Oncolysis Differs Between B-cell Malignancies and Is Not Always Enhanced by GCSF

    doi: 10.1038/mt.2015.149

    Figure Lengend Snippet: Construction and characterization of MV-expressing hGCSF . ( a ) Schematic showing cloning of DNA encoding human cytokine granulocyte colony-stimulating factor (hGCSF) p (+) MV-NSe upstream of M, using AatII and MluI restriction enzymes. ( b ) One step growth

    Article Snippet: It was then PCR amplified with the following primers, forward primer MluI+hGCSF: 5′- agtattac acgcgt atggctggacctgccacccagagc-3′ and reverse primer: AatII_hGCSF: 5′-tacagtcg gacgtc attcagggctgggcaaggtggcg-3′, and the PCR product was cloned into the MVNSe backbone using AatII and MluI restriction endonucleases (New England Biolab, UK), replacing GFP, upstream of MV M gene.

    Techniques: Expressing, Clone Assay

    TNF-α/isoproterenol cotreatment induces chromatin remodelling and histone H3 modifications at the IL-6 promoter in C2C12 myotubes. (A) Schematic representation of the localisation of CREB- and NF-κB-responsive elements in the IL-6 promoter. Relative position of the recognition sites for AatII and HincII and primers used in the restriction enzyme accessibility assay via Real Time PCR (CHART-PCR) are indicated. (B) TNF/iso cotreatment promotes accessibility of the IL-6 promoter. C2C12 cells were treated for 2 hours with veh, iso and/or TNF. Chromatin opening of the promoter region was determined by CHART-PCR as detailed in Materials and Methods . Results represent average ± SD of three independent experiments. (C) Effect of TSA, a histone deacetylase inhibitor, on IL-6 transcription. C2C12 cells were treated for 2 hours with combinations of veh, TSA, iso and/or TNF. mRNA levels of IL-6 were determined via RT-qPCR. Results represent average ± SD of three independent experiments. (D) TNF/iso cotreatment enhances phosphorylation of histone H3 at the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Phosphorylation of histone H3 at serine 10 was determined via ChIP. Results represent average ± SD of three independent experiments. (E-H) Effect of TNF and/or iso treatment on the recruitment of NF-κB, CREB, CBP and RNA polymerase II to the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Recruitment of NF-κB p65, CREB, CBP and RNA polymerase II was measured via ChIP. Results represent average ± SD of three independent experiments. (*) Significantly different from veh. (**) Significantly different from TNF. (***) Significantly different from iso.

    Journal: PLoS ONE

    Article Title: ?-Agonists Selectively Modulate Proinflammatory Gene Expression in Skeletal Muscle Cells via Non-Canonical Nuclear Crosstalk Mechanisms

    doi: 10.1371/journal.pone.0090649

    Figure Lengend Snippet: TNF-α/isoproterenol cotreatment induces chromatin remodelling and histone H3 modifications at the IL-6 promoter in C2C12 myotubes. (A) Schematic representation of the localisation of CREB- and NF-κB-responsive elements in the IL-6 promoter. Relative position of the recognition sites for AatII and HincII and primers used in the restriction enzyme accessibility assay via Real Time PCR (CHART-PCR) are indicated. (B) TNF/iso cotreatment promotes accessibility of the IL-6 promoter. C2C12 cells were treated for 2 hours with veh, iso and/or TNF. Chromatin opening of the promoter region was determined by CHART-PCR as detailed in Materials and Methods . Results represent average ± SD of three independent experiments. (C) Effect of TSA, a histone deacetylase inhibitor, on IL-6 transcription. C2C12 cells were treated for 2 hours with combinations of veh, TSA, iso and/or TNF. mRNA levels of IL-6 were determined via RT-qPCR. Results represent average ± SD of three independent experiments. (D) TNF/iso cotreatment enhances phosphorylation of histone H3 at the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Phosphorylation of histone H3 at serine 10 was determined via ChIP. Results represent average ± SD of three independent experiments. (E-H) Effect of TNF and/or iso treatment on the recruitment of NF-κB, CREB, CBP and RNA polymerase II to the IL-6 promoter. C2C12 cells were treated with veh, iso and/or TNF for 2 hours. Recruitment of NF-κB p65, CREB, CBP and RNA polymerase II was measured via ChIP. Results represent average ± SD of three independent experiments. (*) Significantly different from veh. (**) Significantly different from TNF. (***) Significantly different from iso.

    Article Snippet: AatII and HincII restriction enzymes were obtained from New England BioLabs (Ipswich, MA, USA).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Histone Deacetylase Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation

    Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Journal: Nucleic Acids Research

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    doi: 10.1093/nar/gkl179

    Figure Lengend Snippet: Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Article Snippet: To create linear fragments of differing sizes containing a single Tnp recognition ES, pWSR6103 was digested with PflMI and either XmnI, AatII, NdeI, NarI, BglI or PvuII (all NEB).

    Techniques: Plasmid Preparation, In Vitro, Incubation, Labeling, Agarose Gel Electrophoresis