lambda phage dna  (New England Biolabs)


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    Structured Review

    New England Biolabs lambda phage dna
    Assessment of 5-hmC labeling efficiency. <t>Lambda</t> <t>DNA</t> was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).
    Lambda Phage Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lambda phage dna/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    lambda phage dna - by Bioz Stars, 2020-05
    99/100 stars

    Images

    1) Product Images from "Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays"

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays

    Journal: ACS Nano

    doi: 10.1021/acsnano.8b03023

    Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).
    Figure Legend Snippet: Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Techniques Used: Labeling, Lambda DNA Preparation

    2) Product Images from "Conserved linear dynamics of single-molecule Brownian motion"

    Article Title: Conserved linear dynamics of single-molecule Brownian motion

    Journal: Nature Communications

    doi: 10.1038/ncomms15675

    Lattice occupancy analysis of lambda DNA. ( a ) MSD-Δ t profile of lambda DNA (Δ t =6.4 ms). The red line shows the theoretical MSD-Δ t profile. ( b ) Frequency histogram of the HE-1Δ t distribution of the experimental (top) and the S r A r simulated replicates (bottom) of lambda DNA. ( c ) Averaged MSD-Δ t profiles of the sub-trajectories captured in the same way as in Fig. 7a (red) and Fig. 7b (blue). The red and blue lines are the theoretical MSD-Δ t profiles. The green line shows the overall, theoretical MSD-Δ t profile of the experimental replicates.
    Figure Legend Snippet: Lattice occupancy analysis of lambda DNA. ( a ) MSD-Δ t profile of lambda DNA (Δ t =6.4 ms). The red line shows the theoretical MSD-Δ t profile. ( b ) Frequency histogram of the HE-1Δ t distribution of the experimental (top) and the S r A r simulated replicates (bottom) of lambda DNA. ( c ) Averaged MSD-Δ t profiles of the sub-trajectories captured in the same way as in Fig. 7a (red) and Fig. 7b (blue). The red and blue lines are the theoretical MSD-Δ t profiles. The green line shows the overall, theoretical MSD-Δ t profile of the experimental replicates.

    Techniques Used: Lambda DNA Preparation, Mass Spectrometry

    3) Product Images from "Development of single-cell array for large-scale DNA fluorescence in situ hybridization"

    Article Title: Development of single-cell array for large-scale DNA fluorescence in situ hybridization

    Journal: Lab on a chip

    doi: 10.1039/c2lc40364a

    (a) Phase-contrast micrograph of 10-μm-wide circular PVA dots (black) printed on an APTES-coated glass slide. (b) Height atomic-force microscopy image of a PVA dot on APTES (upper panel) and its line profile (lower panel). Fluorescence micrograph of APTES islands stained with fluorescent rhodamine-B-isothiocyanate (RITC) (c) and YOYO-1–labelled lambda DNA (d).
    Figure Legend Snippet: (a) Phase-contrast micrograph of 10-μm-wide circular PVA dots (black) printed on an APTES-coated glass slide. (b) Height atomic-force microscopy image of a PVA dot on APTES (upper panel) and its line profile (lower panel). Fluorescence micrograph of APTES islands stained with fluorescent rhodamine-B-isothiocyanate (RITC) (c) and YOYO-1–labelled lambda DNA (d).

    Techniques Used: Microscopy, Fluorescence, Staining, Lambda DNA Preparation

    4) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    5) Product Images from "Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays"

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays

    Journal: ACS Nano

    doi: 10.1021/acsnano.8b03023

    Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).
    Figure Legend Snippet: Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Techniques Used: Labeling, Lambda DNA Preparation

    6) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    7) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    8) Product Images from "Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification"

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028661

    Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.
    Figure Legend Snippet: Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.

    Techniques Used: Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Amplification

    9) Product Images from "Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification"

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028661

    Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.
    Figure Legend Snippet: Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.

    Techniques Used: Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Amplification

    Related Articles

    In Vitro:

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
    Article Snippet: .. In vitro methylation of lambda DNA Fully methylated bacteriophage lambda (λ) DNA was acquired by incubating 1 μg of λ DNA in a 20-μl reaction containing 1 U of M.SssI methylase, 1X NEBuffer 2, and freshly-prepared 160 μM S-adenosylmethionine at 37°C for 1 h, followed by heat inactivation of the enzyme at 65°C for 20 min ( ). ..

    Methylation:

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
    Article Snippet: .. In vitro methylation of lambda DNA Fully methylated bacteriophage lambda (λ) DNA was acquired by incubating 1 μg of λ DNA in a 20-μl reaction containing 1 U of M.SssI methylase, 1X NEBuffer 2, and freshly-prepared 160 μM S-adenosylmethionine at 37°C for 1 h, followed by heat inactivation of the enzyme at 65°C for 20 min ( ). ..

    Lambda DNA Preparation:

    Article Title: Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution
    Article Snippet: .. Lambda DNA, HindIII digested Lambda DNA, 1 kb DNA Ladder, and Supercoiled DNA Ladder (all from New England Biolabs, Inc.) were used as double stranded DNA samples. .. Staining was performed at 5 or 10 ng/μL total dsDNA concentration and 1 μM TOTO-3 Iodide (Life Technologies) for at least 1 hour in the dark.

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion
    Article Snippet: .. In vitro methylation of lambda DNA Fully methylated bacteriophage lambda (λ) DNA was acquired by incubating 1 μg of λ DNA in a 20-μl reaction containing 1 U of M.SssI methylase, 1X NEBuffer 2, and freshly-prepared 160 μM S-adenosylmethionine at 37°C for 1 h, followed by heat inactivation of the enzyme at 65°C for 20 min ( ). ..

    Purification:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

    Incubation:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

    Modification:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

    Recombinant:

    Article Title: Efficient modification of λ-DNA substrates for single-molecule studies
    Article Snippet: .. Inserting synthetic oligonucleotides into λ-DNA Recombinant λ-DNA was obtained from strain IF189, which was modified and purified as described above, and 25 μg of the DNA was incubated with 150 U of Nt.BspQI (NEB# R0644S) in a 250 µL reaction with 1X buffer 3.1 (NEB #B7203) at 55 °C for 1 hour. .. The reaction was halted with 1 U of proteinase K (NEB #P8107S) for 1 hour at 55 °C.

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  • 99
    New England Biolabs lambda dna fully methylated bacteriophage lambda
    The workflow of the methyl-sensitive fluorescence polarization (MSFP) assay. <t>Lambda</t> (λ) and human genomic <t>DNA</t> are restricted by MspI (white) or by HpaII each alone or by a combination of HpaII and HpyCH4IV (grey) (Step 1). Subsequently, digested DNA with CpG overhangs at the 5' termini are terminally extended with fluorescence-labelled TAMRA-dCTP (C*, *C) (Step 2). TAMRA-dCTP incorporated into DNA is quantified by fluorescence polarization directly on the plate without additional purification procedures.
    Lambda Dna Fully Methylated Bacteriophage Lambda, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lambda dna fully methylated bacteriophage lambda/product/New England Biolabs
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    lambda dna fully methylated bacteriophage lambda - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

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    The workflow of the methyl-sensitive fluorescence polarization (MSFP) assay. Lambda (λ) and human genomic DNA are restricted by MspI (white) or by HpaII each alone or by a combination of HpaII and HpyCH4IV (grey) (Step 1). Subsequently, digested DNA with CpG overhangs at the 5' termini are terminally extended with fluorescence-labelled TAMRA-dCTP (C*, *C) (Step 2). TAMRA-dCTP incorporated into DNA is quantified by fluorescence polarization directly on the plate without additional purification procedures.

    Journal: PLoS ONE

    Article Title: High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion

    doi: 10.1371/journal.pone.0163184

    Figure Lengend Snippet: The workflow of the methyl-sensitive fluorescence polarization (MSFP) assay. Lambda (λ) and human genomic DNA are restricted by MspI (white) or by HpaII each alone or by a combination of HpaII and HpyCH4IV (grey) (Step 1). Subsequently, digested DNA with CpG overhangs at the 5' termini are terminally extended with fluorescence-labelled TAMRA-dCTP (C*, *C) (Step 2). TAMRA-dCTP incorporated into DNA is quantified by fluorescence polarization directly on the plate without additional purification procedures.

    Article Snippet: In vitro methylation of lambda DNA Fully methylated bacteriophage lambda (λ) DNA was acquired by incubating 1 μg of λ DNA in a 20-μl reaction containing 1 U of M.SssI methylase, 1X NEBuffer 2, and freshly-prepared 160 μM S-adenosylmethionine at 37°C for 1 h, followed by heat inactivation of the enzyme at 65°C for 20 min ( ).

    Techniques: Fluorescence, Purification

    Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Journal: ACS Nano

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays

    doi: 10.1021/acsnano.8b03023

    Figure Lengend Snippet: Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Article Snippet: For the nicking reaction, 900 ng of lambda phage DNA (New England Biolabs) was digested with 30 units of Nt.BspQI nicking enzyme (New England Biolabs) for 2 h at 50 °C in the presence of 3 μL of 10× buffer 3.1 (New England Biolabs) and ultrapure water to a total volume of 30 μL.

    Techniques: Labeling, Lambda DNA Preparation

    Components of the Platform ACE System. ( A ) Platform ACE is a murine artificial chromosome pre-engineered to contain multiple recombination acceptor att P sites. ( B ) ACE Integrase is based on the enzyme lambda integrase, which has been modified as described in the text. The modification renders the integrase functionally independent of bacterial host cell factors and capable of operating in a mammalian context. In the Platform ACE system, an ACE Integrase expression vector is co-transfected with a targeting vector (see below) into a cell line harboring the Platform ACE. The transiently expressed ACE Integrase then catalyses the integration of the targeting vector onto the Platform ACE. ( C ) The ATV is a plasmid-based shuttle vector that conveys a gene(s) of interest onto the Platform ACE by means of targeted recombination between the recombination acceptor att P sites present on the Platform ACE and the recombination donor att B site of the ATV, catalyzed by the ACE Integrase. The presence of a promoterless antibiotic resistance gene downstream of the att B donor site allows for selection of targeted integration events. ( D ) A representation of a ‘loaded’ recombination acceptor site on the Platform ACE. Note that the targeted integration of the targeting vector has resulted in the activation of the promoterless antibiotic resistance gene by virtue of its in frame insertion downstream of the SV40 promoter present in the ACE acceptor site. ( E ) Southern-blot analysis of Platform ACE. Genomic DNA was hybridized with a labeled probe encoding the SV40 promoter and att P site. Lanes 1–3: copy number controls: 50 copies (lane 1), 100 copies (lane 2), 250 copies (lane 3); LMTK − negative control (without ACE, Lane 4) and CHR1 cell line (containing Platform ACE, Lane 5). See Materials and Methods for details.

    Journal: Nucleic Acids Research

    Article Title: A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

    doi: 10.1093/nar/gnh169

    Figure Lengend Snippet: Components of the Platform ACE System. ( A ) Platform ACE is a murine artificial chromosome pre-engineered to contain multiple recombination acceptor att P sites. ( B ) ACE Integrase is based on the enzyme lambda integrase, which has been modified as described in the text. The modification renders the integrase functionally independent of bacterial host cell factors and capable of operating in a mammalian context. In the Platform ACE system, an ACE Integrase expression vector is co-transfected with a targeting vector (see below) into a cell line harboring the Platform ACE. The transiently expressed ACE Integrase then catalyses the integration of the targeting vector onto the Platform ACE. ( C ) The ATV is a plasmid-based shuttle vector that conveys a gene(s) of interest onto the Platform ACE by means of targeted recombination between the recombination acceptor att P sites present on the Platform ACE and the recombination donor att B site of the ATV, catalyzed by the ACE Integrase. The presence of a promoterless antibiotic resistance gene downstream of the att B donor site allows for selection of targeted integration events. ( D ) A representation of a ‘loaded’ recombination acceptor site on the Platform ACE. Note that the targeted integration of the targeting vector has resulted in the activation of the promoterless antibiotic resistance gene by virtue of its in frame insertion downstream of the SV40 promoter present in the ACE acceptor site. ( E ) Southern-blot analysis of Platform ACE. Genomic DNA was hybridized with a labeled probe encoding the SV40 promoter and att P site. Lanes 1–3: copy number controls: 50 copies (lane 1), 100 copies (lane 2), 250 copies (lane 3); LMTK − negative control (without ACE, Lane 4) and CHR1 cell line (containing Platform ACE, Lane 5). See Materials and Methods for details.

    Article Snippet: The lambda integrase gene was amplified by PCR from bacteriophage lambda DNA (cI857 ind 1 Sam 7, New England Biolabs) using the LamInt primer pair (see Supplementary Material, Primer Table.pdf).

    Techniques: Modification, Expressing, Plasmid Preparation, Transfection, Selection, Activation Assay, Southern Blot, Labeling, Negative Control

    DNA extension versus time of λ-DNA treated with Pt(R,R-DACH) or Pt(S,S-DACH). Each data curve was the average of at least three independent measurements.

    Journal: PLoS ONE

    Article Title: Oxaliplatin and Its Enantiomer Induce Different Condensation Dynamics of Single DNA Molecules

    doi: 10.1371/journal.pone.0071556

    Figure Lengend Snippet: DNA extension versus time of λ-DNA treated with Pt(R,R-DACH) or Pt(S,S-DACH). Each data curve was the average of at least three independent measurements.

    Article Snippet: λ-DNA Preparation for Magnetic Tweezers Study The bacteriophage λ-DNA (New England Biolabs), which has two 12-nt cohesive termini, was separately annealed with two 12-nt labeled oligomers (labeled by biotin and digoxigenin, respectively).

    Techniques:

    Force-extension curves and persistence length as a function of incubation time. (A) The force-extension curves of λ-DNA without drug ( L = 16.5±0.04 µm, P = 44.0±2.1 nm), with 60 µM Pt(S,S-DACH) incubated for 3 h ( L = 17.3±0.08 µm, P = 16.5±1.0 nm ) and 60 µM Pt(R,R-DACH) incubated for 3 h ( L = 17.1±0.09 µm, P = 12.9±0.7 nm ). The data were fitted by the WLC model (Eq. 1). (B) The persistence lengths of λ-DNA treated with 60 µM Pt(R,R-DACH) or Pt(S,S-DACH) for different incubation times. Each data point was the mean of twenty independent measurements. The error bars corresponded to 95% confidence intervals. The difference was considered statistically significant when the p value of two-sample t-test was less than 0.05.

    Journal: PLoS ONE

    Article Title: Oxaliplatin and Its Enantiomer Induce Different Condensation Dynamics of Single DNA Molecules

    doi: 10.1371/journal.pone.0071556

    Figure Lengend Snippet: Force-extension curves and persistence length as a function of incubation time. (A) The force-extension curves of λ-DNA without drug ( L = 16.5±0.04 µm, P = 44.0±2.1 nm), with 60 µM Pt(S,S-DACH) incubated for 3 h ( L = 17.3±0.08 µm, P = 16.5±1.0 nm ) and 60 µM Pt(R,R-DACH) incubated for 3 h ( L = 17.1±0.09 µm, P = 12.9±0.7 nm ). The data were fitted by the WLC model (Eq. 1). (B) The persistence lengths of λ-DNA treated with 60 µM Pt(R,R-DACH) or Pt(S,S-DACH) for different incubation times. Each data point was the mean of twenty independent measurements. The error bars corresponded to 95% confidence intervals. The difference was considered statistically significant when the p value of two-sample t-test was less than 0.05.

    Article Snippet: λ-DNA Preparation for Magnetic Tweezers Study The bacteriophage λ-DNA (New England Biolabs), which has two 12-nt cohesive termini, was separately annealed with two 12-nt labeled oligomers (labeled by biotin and digoxigenin, respectively).

    Techniques: Incubation