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  • 99
    New England Biolabs λ dna preparation λ dna
    TAMRA-pPy-stained <t>λ</t> DNA tethered on the surface of a flow cell. ( A ) Free-floating λ DNA (48.5 kb) molecules with a mushroom-like conformation in the flow off condition. ( B ) Fully elongated λ DNA molecules with a flow of 100 μl/min. Arrows indicate flow direction. Scale bar 10 μm. ( C ) Comparison of experimentally measured fluorescence intensity (red) with in silico sequence frequencies from the λ genome sequence. The black solid line represents A/T (W), the gray dotted line indicates four consecutive A/T ( W 4 ), and the grey solid line is W 9 . ( D ) Cross-correlation ( cc ) coefficient values calculated from the alignment of 20 molecular images with the genome using three kinds of binding sequences ( W, W 4 and W 9 ). The control cc was obtained from 100 computer-generated random sequences (*** P
    λ Dna Preparation λ Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mature λ dna
    Bisulfite sanger sequencing of the <t>λ-DNA</t> methylation controls. A section of the sequencing trace for all spikes is presented. The reference sequence and the in-silico converted sequences are shown on top. Green arrows indicate non-CpG cytosines (blue) that are all converted to thymines (red) in all spikes. Orange arrows indicate CpG cytosines that are fully converted in spike 1, partially converted in spikes 2 and 3 and not converted in spike 4.
    Mature λ Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore λ dna
    Fractionation of Hela nuclear extracts. A Schematic representation of a DNA hemicatenane. In this scheme, the two double stranded DNAs that are topologically linked by a hemicatenane are not homologous and therefore are represented by two different colors: blue and red. The light and dark blue strands are complementary, as the light and dark red strands. B Scheme of the procedure of fractionation of Hela nuclear extracts. The procedure starts with an ammonium sulfate precipitation that is followed by four chromatographies on different media, as indicated. C Proteins contained in 1 μL of fraction A10 of the size exclusion chromatography were incubated with radiolabeled dsMC09 (lanes 1–4, 9) or HC (lanes 5–8, 10) in the presence of increasing amount of <t>λ</t> DNA (0 (lanes 4 and 8), 5 (lanes 3 and 7), 10 (lanes 2 and 6), 30 (lanes 1 and 5) ng). After incubation, species were resolved by electrophoresis on a native polyacrylamide gel. Free DNA (dsMC09 or HC) and Protein-HC complexes are indicated. D Analysis of the fraction A10 of the size exclusion chromatography by electrophoresis on a polyacrylamide gel followed by a silver staining of the gel. Lane 1: MW; the size of the proteins is given in kDa; lane2: 30 μL of fraction A10.
    λ Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore intact λ dna
    Trajectory of the center of mass of a <t>λ-DNA</t> molecule at 10 V/cm. The λ-DNA is highly extended and does not always have enough time for complete relaxation before next collision, which for this particular trajectory results in reptation through the 2 nd nanofence array. Five images of a λ-DNA during migration in the nanofence array are included, with the direction of motion being from left-to-right. The DNA moves at constant free solution migration velocity before and after collisions (blue dotted lines). Scale bars = 10 μ m.
    Intact λ Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore λ phage dna
    Fluorescence intensity and applied electrical potential vs time during electrokinetic injection of 5 μg∕mL YOYO-1 labeled <t>λ-phage</t> <t>DNA</t> in 40 mM TAE buffer across a single 300 nm diam×10 μm long PDMS nanopore.
    λ Phage Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa λ dna
    ( A ) Electrophoresis of MSTP-based telomerase reaction products in the absence or presence of HeLa cells lysates, Mg 2+ , and EDTA; ( B ) Electrophoresis of PCR-amplified telomerase reaction products synthesized in the presence of various concentrations of λ DNA.
    λ Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega λ dna
    (a) Representative fluorescence intensity profile of an individual YOYO-1 stained <t>λ-DNA</t> molecule after injection (red line) and confinement (blue line) in the plasma modified nanochannel filled with 2X TBE buffer. Complete injection into the nanochannel
    λ Dna, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    TAMRA-pPy-stained λ DNA tethered on the surface of a flow cell. ( A ) Free-floating λ DNA (48.5 kb) molecules with a mushroom-like conformation in the flow off condition. ( B ) Fully elongated λ DNA molecules with a flow of 100 μl/min. Arrows indicate flow direction. Scale bar 10 μm. ( C ) Comparison of experimentally measured fluorescence intensity (red) with in silico sequence frequencies from the λ genome sequence. The black solid line represents A/T (W), the gray dotted line indicates four consecutive A/T ( W 4 ), and the grey solid line is W 9 . ( D ) Cross-correlation ( cc ) coefficient values calculated from the alignment of 20 molecular images with the genome using three kinds of binding sequences ( W, W 4 and W 9 ). The control cc was obtained from 100 computer-generated random sequences (*** P

    Journal: Nucleic Acids Research

    Article Title: TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

    doi: 10.1093/nar/gky531

    Figure Lengend Snippet: TAMRA-pPy-stained λ DNA tethered on the surface of a flow cell. ( A ) Free-floating λ DNA (48.5 kb) molecules with a mushroom-like conformation in the flow off condition. ( B ) Fully elongated λ DNA molecules with a flow of 100 μl/min. Arrows indicate flow direction. Scale bar 10 μm. ( C ) Comparison of experimentally measured fluorescence intensity (red) with in silico sequence frequencies from the λ genome sequence. The black solid line represents A/T (W), the gray dotted line indicates four consecutive A/T ( W 4 ), and the grey solid line is W 9 . ( D ) Cross-correlation ( cc ) coefficient values calculated from the alignment of 20 molecular images with the genome using three kinds of binding sequences ( W, W 4 and W 9 ). The control cc was obtained from 100 computer-generated random sequences (*** P

    Article Snippet: λ DNA preparation λ DNA (NEB) was diluted to 5 ng/μl (0.16 nM, 7.76 μM as base pairs) with 1 × TE and mixed with 70 μM TAMRA-polypyrrole at a 1:1 volume ratio.

    Techniques: Staining, Flow Cytometry, Fluorescence, In Silico, Sequencing, Binding Assay, Generated

    ( A ) TAMRA-pPy-stained polytene chromosomes from D. melanogaster . Polytene chromosome samples were prepared via a conventional acid-wash protocol and exhibit bands and interbands in chromosomes on a fluorescence microscope. ( B ) Representative fluorescent λ DNA images stained with DAPI and TAMRA-polypyrrole. ( C ) Intensity profiles of DAPI and TAMRA-polypyrrole-stained λ DNA. Red line is TAMRA-polypyrrole, and blue line is DAPI staining. ( D ) Intensity contrast ratios for DAPI and TAMRA-polypyrrole. Bars represent the highest values over the lowest values for each DNA molecule. Error bars represent the minimum and maximum values for the images in (B).

    Journal: Nucleic Acids Research

    Article Title: TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

    doi: 10.1093/nar/gky531

    Figure Lengend Snippet: ( A ) TAMRA-pPy-stained polytene chromosomes from D. melanogaster . Polytene chromosome samples were prepared via a conventional acid-wash protocol and exhibit bands and interbands in chromosomes on a fluorescence microscope. ( B ) Representative fluorescent λ DNA images stained with DAPI and TAMRA-polypyrrole. ( C ) Intensity profiles of DAPI and TAMRA-polypyrrole-stained λ DNA. Red line is TAMRA-polypyrrole, and blue line is DAPI staining. ( D ) Intensity contrast ratios for DAPI and TAMRA-polypyrrole. Bars represent the highest values over the lowest values for each DNA molecule. Error bars represent the minimum and maximum values for the images in (B).

    Article Snippet: λ DNA preparation λ DNA (NEB) was diluted to 5 ng/μl (0.16 nM, 7.76 μM as base pairs) with 1 × TE and mixed with 70 μM TAMRA-polypyrrole at a 1:1 volume ratio.

    Techniques: Staining, Fluorescence, Microscopy

    TAMRA-pPy-stained λ DNA electrostatically immobilized on a surface ( A ) λ DNA on a positively charged surface; yellow arrows indicate the genome direction 5′→3′. ( B ) λ concatemers from monomer to heptamer. Gray bars represent the A/T frequency. ( C ) S. cerevisiae chromosome I DNA fragments aligned with the in silico A/T frequency map. Averaged cc value was 0.64 ± 0.14, whereas cc for computer-generated random images was 0.41 ± 0.06 ( P

    Journal: Nucleic Acids Research

    Article Title: TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

    doi: 10.1093/nar/gky531

    Figure Lengend Snippet: TAMRA-pPy-stained λ DNA electrostatically immobilized on a surface ( A ) λ DNA on a positively charged surface; yellow arrows indicate the genome direction 5′→3′. ( B ) λ concatemers from monomer to heptamer. Gray bars represent the A/T frequency. ( C ) S. cerevisiae chromosome I DNA fragments aligned with the in silico A/T frequency map. Averaged cc value was 0.64 ± 0.14, whereas cc for computer-generated random images was 0.41 ± 0.06 ( P

    Article Snippet: λ DNA preparation λ DNA (NEB) was diluted to 5 ng/μl (0.16 nM, 7.76 μM as base pairs) with 1 × TE and mixed with 70 μM TAMRA-polypyrrole at a 1:1 volume ratio.

    Techniques: Staining, In Silico, Generated

    a) Percentage recovery of DNA for the filtration voltage of 12 V at different filtration times for i) λ DNA and ii) 2kbp DNA molecules. Each filtration profile is an average of two filtration cycles. The color coding is the same for both panels.

    Journal: Lab on a chip

    Article Title: Entropic trap purification of long DNA

    doi: 10.1039/c7lc01355h

    Figure Lengend Snippet: a) Percentage recovery of DNA for the filtration voltage of 12 V at different filtration times for i) λ DNA and ii) 2kbp DNA molecules. Each filtration profile is an average of two filtration cycles. The color coding is the same for both panels.

    Article Snippet: Our experiments use two model systems: λ DNA (48.5 kilobase pairs, kbp, New England Biolabs; R g ≈ 750 nm) as a prototypical long DNA molecule and 2 kbp DNA sample (New England Biolabs; R g ≈ 100 nm) as a model short DNA contaminant.

    Techniques: Filtration

    Percentage recovery of DNA for the filtration time of 600 s at different filtration voltages for a) 2 kbp and b) λ DNA molecules. Each filtration profile is an average of two filtration cycles. The color coding is the same for both panels.

    Journal: Lab on a chip

    Article Title: Entropic trap purification of long DNA

    doi: 10.1039/c7lc01355h

    Figure Lengend Snippet: Percentage recovery of DNA for the filtration time of 600 s at different filtration voltages for a) 2 kbp and b) λ DNA molecules. Each filtration profile is an average of two filtration cycles. The color coding is the same for both panels.

    Article Snippet: Our experiments use two model systems: λ DNA (48.5 kilobase pairs, kbp, New England Biolabs; R g ≈ 750 nm) as a prototypical long DNA molecule and 2 kbp DNA sample (New England Biolabs; R g ≈ 100 nm) as a model short DNA contaminant.

    Techniques: Filtration

    Bisulfite sanger sequencing of the λ-DNA methylation controls. A section of the sequencing trace for all spikes is presented. The reference sequence and the in-silico converted sequences are shown on top. Green arrows indicate non-CpG cytosines (blue) that are all converted to thymines (red) in all spikes. Orange arrows indicate CpG cytosines that are fully converted in spike 1, partially converted in spikes 2 and 3 and not converted in spike 4.

    Journal: PLoS ONE

    Article Title: Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    doi: 10.1371/journal.pone.0135058

    Figure Lengend Snippet: Bisulfite sanger sequencing of the λ-DNA methylation controls. A section of the sequencing trace for all spikes is presented. The reference sequence and the in-silico converted sequences are shown on top. Green arrows indicate non-CpG cytosines (blue) that are all converted to thymines (red) in all spikes. Orange arrows indicate CpG cytosines that are fully converted in spike 1, partially converted in spikes 2 and 3 and not converted in spike 4.

    Article Snippet: Spikes preparation and bisulfite treatment Concentrations of the methylated and unmethylated λ-DNA fragments were quantified with the Qubit fluorometer (Invitrogen) as per manufacturer's instructions.

    Techniques: Sequencing, DNA Methylation Assay, In Silico

    Bisulfite amplicon sanger sequencing. Standard curves were generated by sanger methylation quantification of known methylation level controls. The expected percentage of methylation was plotted versus the observed one. Points represent the mean methylation level of the fifteen and eleven CpG sites for the two regions, respectively, as analyzed with Sanger/ESME (blue line = Lambda control 1, red line = Lambda control 2). Both λ-DNA controls were able to fit linear lines with R 2 values as presented. The correlation coefficients are higher in the data generated from the Premium Bisulfite kit (Diagenode) and the MethylEdge Bisulfite Conversion System (Promega). [P value for i) Lambda control 2 (Qiagen) = 0.1309, ii) rest of samples

    Journal: PLoS ONE

    Article Title: Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    doi: 10.1371/journal.pone.0135058

    Figure Lengend Snippet: Bisulfite amplicon sanger sequencing. Standard curves were generated by sanger methylation quantification of known methylation level controls. The expected percentage of methylation was plotted versus the observed one. Points represent the mean methylation level of the fifteen and eleven CpG sites for the two regions, respectively, as analyzed with Sanger/ESME (blue line = Lambda control 1, red line = Lambda control 2). Both λ-DNA controls were able to fit linear lines with R 2 values as presented. The correlation coefficients are higher in the data generated from the Premium Bisulfite kit (Diagenode) and the MethylEdge Bisulfite Conversion System (Promega). [P value for i) Lambda control 2 (Qiagen) = 0.1309, ii) rest of samples

    Article Snippet: Spikes preparation and bisulfite treatment Concentrations of the methylated and unmethylated λ-DNA fragments were quantified with the Qubit fluorometer (Invitrogen) as per manufacturer's instructions.

    Techniques: Amplification, Sequencing, Generated, Methylation

    Methylation level of λ-DNA control as determined by NGS. The methylation level of all the CpG sites of the amplified region of the λ-DNA control 2 is plotted for all the spikes. Shown is the average value of each CpG in each spike as was calculated from 3 technical replicates (methylated λ-DNA 0% = blue, 3% = green, 5% = red, 7% = light blue, 10% = purple, 25% = yellow, 50% = black, 100% = orange). In order to calculate statistical significance of the methylation level of each spike, we created a dataset of average performance for each spike group and we compared these average performance datasets with the 100% unmethylated average performance set using a t-test. It was demonstrated that all the small-percentage spikes showed significantly higher methylation levels than the 0% methylated group [3% (P = 2.704e-11), 5% (P = 1.685e-21), 7% (P = 1.181e-23), 10% (P = 2.991e-19)]. Errors bars = SD

    Journal: PLoS ONE

    Article Title: Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    doi: 10.1371/journal.pone.0135058

    Figure Lengend Snippet: Methylation level of λ-DNA control as determined by NGS. The methylation level of all the CpG sites of the amplified region of the λ-DNA control 2 is plotted for all the spikes. Shown is the average value of each CpG in each spike as was calculated from 3 technical replicates (methylated λ-DNA 0% = blue, 3% = green, 5% = red, 7% = light blue, 10% = purple, 25% = yellow, 50% = black, 100% = orange). In order to calculate statistical significance of the methylation level of each spike, we created a dataset of average performance for each spike group and we compared these average performance datasets with the 100% unmethylated average performance set using a t-test. It was demonstrated that all the small-percentage spikes showed significantly higher methylation levels than the 0% methylated group [3% (P = 2.704e-11), 5% (P = 1.685e-21), 7% (P = 1.181e-23), 10% (P = 2.991e-19)]. Errors bars = SD

    Article Snippet: Spikes preparation and bisulfite treatment Concentrations of the methylated and unmethylated λ-DNA fragments were quantified with the Qubit fluorometer (Invitrogen) as per manufacturer's instructions.

    Techniques: Methylation, Next-Generation Sequencing, Amplification

    DNA titration using SYBR ® Green I and PicoGreen ® dyes . PicoGreen ® fluorescence (A) and SYBR ® Green I fluorescence (B) with bacteriophage λ DNA in absence (black circle, solid line), or presence (dotted lines) of different amounts of detergents: saponin 0.008% + Triton X-100 0.08% (white triangle); saponin 0.008% (white square); Triton X-100 0.08% (white diamonds); Triton X-100 2% (white circle). Background fluorescence, defined as fluorescence detected in the absence of DNA, was subtracted from each data point. Fluorescence is measured as arbitrary units (AU). Data show average from three replicate experiments. Error bars indicate standard deviations. Lines were calculated by linear regression; r 2 > 0.99.

    Journal: Malaria Journal

    Article Title: Haemoglobin interference and increased sensitivity of fluorimetric assays for quantification of low-parasitaemia Plasmodium infected erythrocytes

    doi: 10.1186/1475-2875-8-279

    Figure Lengend Snippet: DNA titration using SYBR ® Green I and PicoGreen ® dyes . PicoGreen ® fluorescence (A) and SYBR ® Green I fluorescence (B) with bacteriophage λ DNA in absence (black circle, solid line), or presence (dotted lines) of different amounts of detergents: saponin 0.008% + Triton X-100 0.08% (white triangle); saponin 0.008% (white square); Triton X-100 0.08% (white diamonds); Triton X-100 2% (white circle). Background fluorescence, defined as fluorescence detected in the absence of DNA, was subtracted from each data point. Fluorescence is measured as arbitrary units (AU). Data show average from three replicate experiments. Error bars indicate standard deviations. Lines were calculated by linear regression; r 2 > 0.99.

    Article Snippet: Standard curves of DNA were performed by diluting bacteriophage λ DNA, provided at 100 μg/mL in the Quant-iT™ PicoGreen® Kits (Molecular Probes™, Invitrogen) with DNase-free water.

    Techniques: Titration, SYBR Green Assay, Fluorescence

    The construction of mismatched DNA used in single-molecule total internal reflection fluorescence (smTIRF) microscopy a , A schematic illustration for the construction of a 17.3-kb mismatched DNA. L or R (blue) indicates the orientation of the DNA relative to the L and R cos end of λ-phage DNA. P (red) indicates the 5′-phosphate of the DNA. b , A schematic illustration of 17.3-kb mismatched DNA observation by prism-based smTIRF microscopy. c , Representative mismatched DNA visualized by smTIRF microscopy in the absence of flow. The DNA was stained with Sytox Orange and a 40 × 85 µm field of view is shown. d , A schematic illustration of the DNA length determination. e , The length distribution of the mismatched DNA observed by smTIRF microscopy. Gaussian fit of the data are shown along with the mean ± s.d.

    Journal: Nature

    Article Title: Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair

    doi: 10.1038/nature20562

    Figure Lengend Snippet: The construction of mismatched DNA used in single-molecule total internal reflection fluorescence (smTIRF) microscopy a , A schematic illustration for the construction of a 17.3-kb mismatched DNA. L or R (blue) indicates the orientation of the DNA relative to the L and R cos end of λ-phage DNA. P (red) indicates the 5′-phosphate of the DNA. b , A schematic illustration of 17.3-kb mismatched DNA observation by prism-based smTIRF microscopy. c , Representative mismatched DNA visualized by smTIRF microscopy in the absence of flow. The DNA was stained with Sytox Orange and a 40 × 85 µm field of view is shown. d , A schematic illustration of the DNA length determination. e , The length distribution of the mismatched DNA observed by smTIRF microscopy. Gaussian fit of the data are shown along with the mean ± s.d.

    Article Snippet: λ-phage DNA (3.2 nM, Thermo Scientific) was ligated with the lambda mismatch 1 oligonucleotide (800 nM; , ) at room temperature (22 °C) overnight.

    Techniques: Fluorescence, Microscopy, Flow Cytometry, Staining

    Fractionation of Hela nuclear extracts. A Schematic representation of a DNA hemicatenane. In this scheme, the two double stranded DNAs that are topologically linked by a hemicatenane are not homologous and therefore are represented by two different colors: blue and red. The light and dark blue strands are complementary, as the light and dark red strands. B Scheme of the procedure of fractionation of Hela nuclear extracts. The procedure starts with an ammonium sulfate precipitation that is followed by four chromatographies on different media, as indicated. C Proteins contained in 1 μL of fraction A10 of the size exclusion chromatography were incubated with radiolabeled dsMC09 (lanes 1–4, 9) or HC (lanes 5–8, 10) in the presence of increasing amount of λ DNA (0 (lanes 4 and 8), 5 (lanes 3 and 7), 10 (lanes 2 and 6), 30 (lanes 1 and 5) ng). After incubation, species were resolved by electrophoresis on a native polyacrylamide gel. Free DNA (dsMC09 or HC) and Protein-HC complexes are indicated. D Analysis of the fraction A10 of the size exclusion chromatography by electrophoresis on a polyacrylamide gel followed by a silver staining of the gel. Lane 1: MW; the size of the proteins is given in kDa; lane2: 30 μL of fraction A10.

    Journal: bioRxiv

    Article Title: Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

    doi: 10.1101/844126

    Figure Lengend Snippet: Fractionation of Hela nuclear extracts. A Schematic representation of a DNA hemicatenane. In this scheme, the two double stranded DNAs that are topologically linked by a hemicatenane are not homologous and therefore are represented by two different colors: blue and red. The light and dark blue strands are complementary, as the light and dark red strands. B Scheme of the procedure of fractionation of Hela nuclear extracts. The procedure starts with an ammonium sulfate precipitation that is followed by four chromatographies on different media, as indicated. C Proteins contained in 1 μL of fraction A10 of the size exclusion chromatography were incubated with radiolabeled dsMC09 (lanes 1–4, 9) or HC (lanes 5–8, 10) in the presence of increasing amount of λ DNA (0 (lanes 4 and 8), 5 (lanes 3 and 7), 10 (lanes 2 and 6), 30 (lanes 1 and 5) ng). After incubation, species were resolved by electrophoresis on a native polyacrylamide gel. Free DNA (dsMC09 or HC) and Protein-HC complexes are indicated. D Analysis of the fraction A10 of the size exclusion chromatography by electrophoresis on a polyacrylamide gel followed by a silver staining of the gel. Lane 1: MW; the size of the proteins is given in kDa; lane2: 30 μL of fraction A10.

    Article Snippet: All columns were prepared as recommended by the manufacturer and equilibrated in the indicated loading buffer before use. λ DNA was from Sigma.

    Techniques: Fractionation, Size-exclusion Chromatography, Incubation, Electrophoresis, Silver Staining

    Identification of HC-specific binding proteins in the fractions A10 and A11 of the size exclusion chromatography. A Products of the interaction between radiolabeled HC (16 femtomoles) and proteins of the fraction A10 of the size exclusion chromatography (45 μL) were resolved by electrophoresis on a polyacrylamide gel (lanes 3, 5, 8, 10). 12 (lanes 2, 3, 7, 8) or 23 (lanes 4, 5, 9, 10) ng of λ DNA were added to reduce non specific binding. Sample without protein (lanes 1 and 6) or without HC (lanes 2, 4, 7, 9) were also loaded to serve as control for mass spectrometry analysis. Free DNA (dsMC or HC) and Protein-HC complexes are indicated. On the right panel, the gel pieces that were analyzed by mass spectrometry are shown as green rectangles and the name of the gel pieces of interest is indicated on the right side of the figure. B Same as in (A) but with fraction A11 of the size exclusion chromatography. No λ DNA was added in the reaction mixes. Lanes 1 and 4: HC without fraction A11; lanes 2 and 5: fraction A11 without HC; lanes 3 and 6: HC + fraction A11. On the right panel, the green rectangles represent the two gel pieces that were analyzed by mass spectrometry. The name of the gel piece of interest is indicated on the right side of the figure. C Fractions A10 and A11 were tested for their content of PSPC1, SND1, SFPQ and PTBP1 proteins by western blot. Lanes 1, 4, 7, 10: MW; the size of the proteins is given in kDa; lanes 2, 5, 8, 11: fraction A10; lanes 3, 6, 9, 12: fraction A11. The identity of the antibodies used to probe the faction is indicated above each panel. The arrow points to the protein of interest. D and E Three samples were prepared and centrifuged on a sucrose gradient: sample with HC, sample with fraction A10 and sample with fraction A10 mixed with HC. After centrifugation, 200 μL fractions were collected from the top to the bottom of the gradient. Number of the fraction increases from top to bottom. In (D), fractions 5 to 10 of the sucrose gradients were tested for their SND1 content by western blot (band indicated by an arrow on the right side of the panel). Lanes 1, 3, 5, 7, 9, 11: the fraction A10 was loaded on the sucrose gradient. Lanes 2, 4, 6, 8, 10, 12: the mixture (fraction A10 + HC) was loaded on the sucrose gradient. Bands on the membrane were quantified using Image J software and the relative intensity of each band is plotted as a function of fraction. The white bars correspond to the fraction A10 sample and the hatched bars to the (fraction A10 + HC) sample. In (E), fractions 5 to 10 of the sucrose gradients were tested for their radioactivity content. An aliquot of each fraction (0.5 μL) was spotted on a nitrocellulose membrane. When dried, the membrane was exposed on a 32 P-sensitive screen. After exposure, the screen was scanned. The radioactivity profile of the HC sample is compared with that of the (fraction A10 + HC) sample.

    Journal: bioRxiv

    Article Title: Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

    doi: 10.1101/844126

    Figure Lengend Snippet: Identification of HC-specific binding proteins in the fractions A10 and A11 of the size exclusion chromatography. A Products of the interaction between radiolabeled HC (16 femtomoles) and proteins of the fraction A10 of the size exclusion chromatography (45 μL) were resolved by electrophoresis on a polyacrylamide gel (lanes 3, 5, 8, 10). 12 (lanes 2, 3, 7, 8) or 23 (lanes 4, 5, 9, 10) ng of λ DNA were added to reduce non specific binding. Sample without protein (lanes 1 and 6) or without HC (lanes 2, 4, 7, 9) were also loaded to serve as control for mass spectrometry analysis. Free DNA (dsMC or HC) and Protein-HC complexes are indicated. On the right panel, the gel pieces that were analyzed by mass spectrometry are shown as green rectangles and the name of the gel pieces of interest is indicated on the right side of the figure. B Same as in (A) but with fraction A11 of the size exclusion chromatography. No λ DNA was added in the reaction mixes. Lanes 1 and 4: HC without fraction A11; lanes 2 and 5: fraction A11 without HC; lanes 3 and 6: HC + fraction A11. On the right panel, the green rectangles represent the two gel pieces that were analyzed by mass spectrometry. The name of the gel piece of interest is indicated on the right side of the figure. C Fractions A10 and A11 were tested for their content of PSPC1, SND1, SFPQ and PTBP1 proteins by western blot. Lanes 1, 4, 7, 10: MW; the size of the proteins is given in kDa; lanes 2, 5, 8, 11: fraction A10; lanes 3, 6, 9, 12: fraction A11. The identity of the antibodies used to probe the faction is indicated above each panel. The arrow points to the protein of interest. D and E Three samples were prepared and centrifuged on a sucrose gradient: sample with HC, sample with fraction A10 and sample with fraction A10 mixed with HC. After centrifugation, 200 μL fractions were collected from the top to the bottom of the gradient. Number of the fraction increases from top to bottom. In (D), fractions 5 to 10 of the sucrose gradients were tested for their SND1 content by western blot (band indicated by an arrow on the right side of the panel). Lanes 1, 3, 5, 7, 9, 11: the fraction A10 was loaded on the sucrose gradient. Lanes 2, 4, 6, 8, 10, 12: the mixture (fraction A10 + HC) was loaded on the sucrose gradient. Bands on the membrane were quantified using Image J software and the relative intensity of each band is plotted as a function of fraction. The white bars correspond to the fraction A10 sample and the hatched bars to the (fraction A10 + HC) sample. In (E), fractions 5 to 10 of the sucrose gradients were tested for their radioactivity content. An aliquot of each fraction (0.5 μL) was spotted on a nitrocellulose membrane. When dried, the membrane was exposed on a 32 P-sensitive screen. After exposure, the screen was scanned. The radioactivity profile of the HC sample is compared with that of the (fraction A10 + HC) sample.

    Article Snippet: All columns were prepared as recommended by the manufacturer and equilibrated in the indicated loading buffer before use. λ DNA was from Sigma.

    Techniques: Binding Assay, Size-exclusion Chromatography, Electrophoresis, Mass Spectrometry, Western Blot, Centrifugation, Software, Radioactivity

    Trajectory of the center of mass of a λ-DNA molecule at 10 V/cm. The λ-DNA is highly extended and does not always have enough time for complete relaxation before next collision, which for this particular trajectory results in reptation through the 2 nd nanofence array. Five images of a λ-DNA during migration in the nanofence array are included, with the direction of motion being from left-to-right. The DNA moves at constant free solution migration velocity before and after collisions (blue dotted lines). Scale bars = 10 μ m.

    Journal: Lab on a chip

    Article Title: DNA electrophoresis in a nanofence array

    doi: 10.1039/c2lc00016d

    Figure Lengend Snippet: Trajectory of the center of mass of a λ-DNA molecule at 10 V/cm. The λ-DNA is highly extended and does not always have enough time for complete relaxation before next collision, which for this particular trajectory results in reptation through the 2 nd nanofence array. Five images of a λ-DNA during migration in the nanofence array are included, with the direction of motion being from left-to-right. The DNA moves at constant free solution migration velocity before and after collisions (blue dotted lines). Scale bars = 10 μ m.

    Article Snippet: Our stock mixture for the DNA separation experiments contains XhoI digest fragments at an approximate concentration of 18 μg/ml and intact λ-DNA at an approximate concentration of 16 μg/ml in 2.2× TBE buffer (196 mM tris base, 196 mM boric acid, 6.2 mM ethylenediaminetetraacetic acid, Sigma).

    Techniques: Migration

    The scattering wave vector, q , dependence of the reduced relaxation rates, Γ red , at 25 °C for quenched (A) and unquenched (B) λ-DNA in solution at 20μg/ml, and the unquenched DNA solutions at 140μg/ml (C). The solid

    Journal: The Journal of Physical Chemistry. a

    Article Title: Mechanistic insights into the Structure and Dynamics of Entangled and Hydrated Lambda-Phage DNA

    doi: 10.1021/jp2108363

    Figure Lengend Snippet: The scattering wave vector, q , dependence of the reduced relaxation rates, Γ red , at 25 °C for quenched (A) and unquenched (B) λ-DNA in solution at 20μg/ml, and the unquenched DNA solutions at 140μg/ml (C). The solid

    Article Snippet: To check whether the investigated DNA solutions did not contain any dust particles that affect the PCS data, a portion of the quenched solution (at 20μg/ml), that is a solution of unentanlged and non-overlapping λ-DNA chains, was filtered with 0.65μm Millipore membrane (Ultrafree-CL) without any external pressure.

    Techniques: Plasmid Preparation

    Typical AFM height images of structures for quenched (A) and unquenched (B) λ-DNA molecules at 0.1μg/ml in solution after immobilization onto APTES-modified muscovite mica sheets. Images were second-order plane-fitted using the Scanning

    Journal: The Journal of Physical Chemistry. a

    Article Title: Mechanistic insights into the Structure and Dynamics of Entangled and Hydrated Lambda-Phage DNA

    doi: 10.1021/jp2108363

    Figure Lengend Snippet: Typical AFM height images of structures for quenched (A) and unquenched (B) λ-DNA molecules at 0.1μg/ml in solution after immobilization onto APTES-modified muscovite mica sheets. Images were second-order plane-fitted using the Scanning

    Article Snippet: To check whether the investigated DNA solutions did not contain any dust particles that affect the PCS data, a portion of the quenched solution (at 20μg/ml), that is a solution of unentanlged and non-overlapping λ-DNA chains, was filtered with 0.65μm Millipore membrane (Ultrafree-CL) without any external pressure.

    Techniques: Modification

    Fluorescence intensity and applied electrical potential vs time during electrokinetic injection of 5 μg∕mL YOYO-1 labeled λ-phage DNA in 40 mM TAE buffer across a single 300 nm diam×10 μm long PDMS nanopore.

    Journal: Biomicrofluidics

    Article Title: Single nanopore transport of synthetic and biological polyelectrolytes in three-dimensional hybrid microfluidic/nanofluidic devices

    doi: 10.1063/1.3059546

    Figure Lengend Snippet: Fluorescence intensity and applied electrical potential vs time during electrokinetic injection of 5 μg∕mL YOYO-1 labeled λ-phage DNA in 40 mM TAE buffer across a single 300 nm diam×10 μm long PDMS nanopore.

    Article Snippet: Polystyrene sulfonate sodium salt (PSS), polyallylamine chloride salt (PAA), tris(hydroxymethyl)aminomethane (TRIS), ethylenediaminetetraacetic (EDTA), λ-phage DNA, and acetic acid (Sigma Aldrich Co., St. Louis, MO) and YOYO-1 (Invitrogen, Carlsbad, CA) were used as received.

    Techniques: Fluorescence, Injection, Labeling

    Localization and transport of lambda phage DNA molecules from a mature mother to its bud. Phase contrast and fluorescence micrographs of a GV labeled with 0.02 mol% TexasRed-DHPE and encapsulated DNA stained by SYBR Green I. The first image was taken 13 min after the mixing of a dispersion of DNA-containing immature mothers, a catalytic solution (copper (I) chloride, 10 mM; ascorbic acid, 20 mM; and deionized water) and reactive precursors LH (9 mM) and AH/Chol (9/1 mM) ( t = 0 sec). Images were acquired at: ( A ) 13:03 sec, ( B ) 18:23 sec, ( C ) 20:01 sec, ( D ) 20:29 sec, ( E ) 22:06 sec, ( F ) 23:13 sec, ( G ) 35:14 sec, ( H ) 38:18 sec. Scale bar, 10 μm.

    Journal: Scientific Reports

    Article Title: Budding and Division of Giant Vesicles Linked to Phospholipid Production

    doi: 10.1038/s41598-018-36183-9

    Figure Lengend Snippet: Localization and transport of lambda phage DNA molecules from a mature mother to its bud. Phase contrast and fluorescence micrographs of a GV labeled with 0.02 mol% TexasRed-DHPE and encapsulated DNA stained by SYBR Green I. The first image was taken 13 min after the mixing of a dispersion of DNA-containing immature mothers, a catalytic solution (copper (I) chloride, 10 mM; ascorbic acid, 20 mM; and deionized water) and reactive precursors LH (9 mM) and AH/Chol (9/1 mM) ( t = 0 sec). Images were acquired at: ( A ) 13:03 sec, ( B ) 18:23 sec, ( C ) 20:01 sec, ( D ) 20:29 sec, ( E ) 22:06 sec, ( F ) 23:13 sec, ( G ) 35:14 sec, ( H ) 38:18 sec. Scale bar, 10 μm.

    Article Snippet: Lambda phage DNA (48502-base pair) methylated from Escherichia coli host strain W3110 was purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Fluorescence, Labeling, Staining, SYBR Green Assay, Size-exclusion Chromatography

    ( A ) Electrophoresis of MSTP-based telomerase reaction products in the absence or presence of HeLa cells lysates, Mg 2+ , and EDTA; ( B ) Electrophoresis of PCR-amplified telomerase reaction products synthesized in the presence of various concentrations of λ DNA.

    Journal: Molecules

    Article Title: A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

    doi: 10.3390/molecules181011751

    Figure Lengend Snippet: ( A ) Electrophoresis of MSTP-based telomerase reaction products in the absence or presence of HeLa cells lysates, Mg 2+ , and EDTA; ( B ) Electrophoresis of PCR-amplified telomerase reaction products synthesized in the presence of various concentrations of λ DNA.

    Article Snippet: Materials and Reagents All deoxyribo-oligonucleotides were high-performance liquid chromatography (HPLC) purification-grade deoxyribo-oligonucleotides, and were purchased from Tsukuba Oligo Service Co., Ltd., (Ibaraki, Japan), or were provided in a TRAPEZE telomerase detection kit from EMD Millipore Corporation (Billerica, MA, USA). λ DNA was purchased from Takara Bio Inc. (Shiga, Japan).

    Techniques: Electrophoresis, Polymerase Chain Reaction, Amplification, Synthesized

    (a) Representative fluorescence intensity profile of an individual YOYO-1 stained λ-DNA molecule after injection (red line) and confinement (blue line) in the plasma modified nanochannel filled with 2X TBE buffer. Complete injection into the nanochannel

    Journal: The Analyst

    Article Title: Surface Charge, Electroosmotic Flow and DNA Extension in Chemically Modified Thermoplastic Nanoslits and Nanochannels

    doi: 10.1039/c4an01439a

    Figure Lengend Snippet: (a) Representative fluorescence intensity profile of an individual YOYO-1 stained λ-DNA molecule after injection (red line) and confinement (blue line) in the plasma modified nanochannel filled with 2X TBE buffer. Complete injection into the nanochannel

    Article Snippet: To study the electrokinetic parameters and extension length of λ-DNA, 100 × 100 nm nanochannels were used. λ-DNA (Promega Corporation) were stained with the bis-intercalating dye, YOYO-1 (Molecular Probes, Eugene, OR) at a base-pair/dye ratio of 5:1 in an electrolyte solution of 1X TBE (89 mM Tris, 89 mM Borate, 1 mM EDTA) with the addition of 4% v/v β-mercaptoethanol as a radical scavenger to minimize photo-induced damage (photobleaching and/or photonicking).

    Techniques: Fluorescence, Staining, Injection, Modification

    Thermodynamic equilibrium of  f–X  measurements. ( A ) Reversibility of HU-λ DNA binding demonstrated via  f–X  curves. DNA extension decreased when HU concentration was increased from 0 to 100 nM, and then to 500 nM. One hour after replacing the 500 nM HU solution with 100 nM HU solution, the  f–X  curve of ‘100 return’ overlaid with the curve previously obtained at 100 nM HU, indicating reversibility of binding. Each experimental data point, presented as the mean value with error bars (SE), was obtained from 15 to 20 separate measurements. The errors range from 6 nm at 8.4 pN to 0.15 µm at 0.05 pN. ( B ) Reversibility of Fis-pFOS1 fragment binding. As the Fis concentration was cycled up and down, the  f–X  curves shifted right and left. The curve of ‘10 return’ was the same as the previous 10 nM curve. Each experimental data point, presented as the mean value with error bars (SE), was obtained from 3700 to 4300 separate fast extension measurements. The errors range from 3 nm at 3.8 pN to 0.03 µm for 0.06 pN.

    Journal: Nucleic Acids Research

    Article Title: Force-driven unbinding of proteins HU and Fis from DNA quantified using a thermodynamic Maxwell relation

    doi: 10.1093/nar/gkr141

    Figure Lengend Snippet: Thermodynamic equilibrium of f–X measurements. ( A ) Reversibility of HU-λ DNA binding demonstrated via f–X curves. DNA extension decreased when HU concentration was increased from 0 to 100 nM, and then to 500 nM. One hour after replacing the 500 nM HU solution with 100 nM HU solution, the f–X curve of ‘100 return’ overlaid with the curve previously obtained at 100 nM HU, indicating reversibility of binding. Each experimental data point, presented as the mean value with error bars (SE), was obtained from 15 to 20 separate measurements. The errors range from 6 nm at 8.4 pN to 0.15 µm at 0.05 pN. ( B ) Reversibility of Fis-pFOS1 fragment binding. As the Fis concentration was cycled up and down, the f–X curves shifted right and left. The curve of ‘10 return’ was the same as the previous 10 nM curve. Each experimental data point, presented as the mean value with error bars (SE), was obtained from 3700 to 4300 separate fast extension measurements. The errors range from 3 nm at 3.8 pN to 0.03 µm for 0.06 pN.

    Article Snippet: The first, 48.5 kb λ DNA (Promega, Madison, WI, USA), was end-labeled with biotin and digoxygenin as described previously ( , ), and was used for studies of HU.

    Techniques: Binding Assay, Concentration Assay

    f–X  curves of protein–DNA complexes. Each experimental data point, presented as the mean value with error bars (SE), was obtained from 6 to 10 separate measurements. Successively higher concentrations led to more protein binding, and consequently more compaction against the applied force.  f–X  curves shifted right as the added protein concentrations were increased. ( A ) HU-concentration dependence of  f–X  curves of a λ DNA. Model calculations (lines) from   Equation (7)  fit the experimental data (dots) well. ( B ) Fis-concentration dependence of  f–X  curve of a pFOS1 fragment.

    Journal: Nucleic Acids Research

    Article Title: Force-driven unbinding of proteins HU and Fis from DNA quantified using a thermodynamic Maxwell relation

    doi: 10.1093/nar/gkr141

    Figure Lengend Snippet: f–X curves of protein–DNA complexes. Each experimental data point, presented as the mean value with error bars (SE), was obtained from 6 to 10 separate measurements. Successively higher concentrations led to more protein binding, and consequently more compaction against the applied force. f–X curves shifted right as the added protein concentrations were increased. ( A ) HU-concentration dependence of f–X curves of a λ DNA. Model calculations (lines) from Equation (7) fit the experimental data (dots) well. ( B ) Fis-concentration dependence of f–X curve of a pFOS1 fragment.

    Article Snippet: The first, 48.5 kb λ DNA (Promega, Madison, WI, USA), was end-labeled with biotin and digoxygenin as described previously ( , ), and was used for studies of HU.

    Techniques: Protein Binding, Concentration Assay

    Changes of protein numbers on a single stretched DNA molecule. For the binding of HU or Fis under conditions where they compacted DNA, proteins were released from DNA as force was increased. ( A ) Changes of HU numbers. In experiments (dots), the loss of proteins on a λ DNA increased as the forces increased from 0.06 to 6.3 pN, and as the HU concentrations increased from 25 to 500 nM. In the model calculation (lines), the loss of proteins increased smoothly and reached a plateau as the forces increased from 0 to 6.3 pN. For 500 nM HU, the loss of proteins number was 520 from model calculation, and 610 from experiments. ( B ) Changes of Fis numbers calculated from experimental data. The loss of proteins on a 6.2 kb pFOS1 fragment increased to 220 dimers for the 20 nM Fis experiment as the forces increased from 0.05 to 12.0 pN. No saturation was observed in the force and concentration ranges.

    Journal: Nucleic Acids Research

    Article Title: Force-driven unbinding of proteins HU and Fis from DNA quantified using a thermodynamic Maxwell relation

    doi: 10.1093/nar/gkr141

    Figure Lengend Snippet: Changes of protein numbers on a single stretched DNA molecule. For the binding of HU or Fis under conditions where they compacted DNA, proteins were released from DNA as force was increased. ( A ) Changes of HU numbers. In experiments (dots), the loss of proteins on a λ DNA increased as the forces increased from 0.06 to 6.3 pN, and as the HU concentrations increased from 25 to 500 nM. In the model calculation (lines), the loss of proteins increased smoothly and reached a plateau as the forces increased from 0 to 6.3 pN. For 500 nM HU, the loss of proteins number was 520 from model calculation, and 610 from experiments. ( B ) Changes of Fis numbers calculated from experimental data. The loss of proteins on a 6.2 kb pFOS1 fragment increased to 220 dimers for the 20 nM Fis experiment as the forces increased from 0.05 to 12.0 pN. No saturation was observed in the force and concentration ranges.

    Article Snippet: The first, 48.5 kb λ DNA (Promega, Madison, WI, USA), was end-labeled with biotin and digoxygenin as described previously ( , ), and was used for studies of HU.

    Techniques: Binding Assay, Concentration Assay

    C. heterostrophus secretes DNases, and secretion is induced by plant tissue. WT, nuc1 mutant, nuc2 mutant, and nuc1 nuc2 double-mutant filtrates degrade intact λ DNA in the presence of plant material. The nuc1 single mutant and nuc1 nuc2 double mutant degrade lambda DNA less well than the WT or the nuc2 single mutant. This indicates that DNase(s) are secreted by the fungus, that the nuc1 mutant secretes a DNase that is important in DNA degradation, and that secretion is induced by host material. Left, size markers in kilobases. +, addition of corn leaf (CL) fragments, lambda DNA, or purified RQ1 RNase-free DNase. Culture filtrates examined were from WT strain C4, nuc1 mutant strains 144206-3-1 and 8-1, nuc2 mutant strains 149183-2-1 and 3-1, and nuc1 nuc2 double-mutant strains 144206/149183-4-1 and 8-1. The negative-control reaction with λ DNA did not degrade the DNA, while the positive-control reaction with λ DNA plus DNase did. Also note that λ DNA was not degraded by the CL material.

    Journal: mBio

    Article Title: A DNase from a Fungal Phytopathogen Is a Virulence Factor Likely Deployed as Counter Defense against Host-Secreted Extracellular DNA

    doi: 10.1128/mBio.02805-18

    Figure Lengend Snippet: C. heterostrophus secretes DNases, and secretion is induced by plant tissue. WT, nuc1 mutant, nuc2 mutant, and nuc1 nuc2 double-mutant filtrates degrade intact λ DNA in the presence of plant material. The nuc1 single mutant and nuc1 nuc2 double mutant degrade lambda DNA less well than the WT or the nuc2 single mutant. This indicates that DNase(s) are secreted by the fungus, that the nuc1 mutant secretes a DNase that is important in DNA degradation, and that secretion is induced by host material. Left, size markers in kilobases. +, addition of corn leaf (CL) fragments, lambda DNA, or purified RQ1 RNase-free DNase. Culture filtrates examined were from WT strain C4, nuc1 mutant strains 144206-3-1 and 8-1, nuc2 mutant strains 149183-2-1 and 3-1, and nuc1 nuc2 double-mutant strains 144206/149183-4-1 and 8-1. The negative-control reaction with λ DNA did not degrade the DNA, while the positive-control reaction with λ DNA plus DNase did. Also note that λ DNA was not degraded by the CL material.

    Article Snippet: Reactions were run on 1% agarose gels at 100 V for 15 min. As a positive control for λ DNA degradation, 1 unit of RQ1 DNase (catalog no. M610A; Promega) was used.

    Techniques: Mutagenesis, Lambda DNA Preparation, Purification, Negative Control, Positive Control