dnase  (Worthington Biochemical)


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    Name:
    Deoxyribonuclease I
    Description:
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    Catalog Number:
    LS002004
    Price:
    33
    Source:
    Bovine Pancreas
    Size:
    5 mg
    Cas Number:
    9003.98.9
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    Structured Review

    Worthington Biochemical dnase
    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the <t>DNA</t> at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with <t>DNase</t> and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    https://www.bioz.com/result/dnase/product/Worthington Biochemical
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase - by Bioz Stars, 2021-10
    98/100 stars

    Images

    1) Product Images from "Initiation-specific alleles of the Cdc45 helicase-activating protein"

    Article Title: Initiation-specific alleles of the Cdc45 helicase-activating protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0214426

    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Tube Formation Assay

    Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Produced, Agarose Gel Electrophoresis, Mutagenesis

    2) Product Images from "Neutrophil extracellular traps in the central nervous system hinder bacterial clearance during pneumococcal meningitis"

    Article Title: Neutrophil extracellular traps in the central nervous system hinder bacterial clearance during pneumococcal meningitis

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09040-0

    DNase facilitates bacterial clearance in a rat model of pneumococcal meningitis. Rats received a subarachnoid infusion of either S. pneumoniae SP001 strain (infected) or equal volume of saline solution (control). a After the rats were sacrificed, the brain, the right lung, and the spleen were collected and homogenized immediately. The blood was collected and centrifuged to obtain plasma. Organ homogenates and blood plasma samples were spread onto agar plates and resulting bacterial colonies were counted after 24 h. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and error bars denote standard deviations. b To determine the effect of intrathecal DNase treatment, infected rats either received a subarachnoid infusion of 10 units of DNase simultaneously (0 h) or 10 h after the infection, or they received an equal volume of saline vehicle solution simultaneously to the infection. To determine the effect of intravenous DNase treatment, infected rats either received an intravenous bolus dose of 3500 units of DNase 6 h after the infection, followed by intravenous infusion of 780 units/h over the next 18 h, or they received an equal volume of saline vehicle control in the same manner. In all cases, uninfected (control) rats received an equal volume of saline vehicle control either intrathecally or intravenously as indicated. All rats were sacrificed 24 h after the infection. Cerebrospinal fluid was collected for visualization of NETs only by immunofluorescence against rat myeloperoxidase (red) and DNA (blue). Areas of red and blue colocalization represent NETs. Scale bars denote 200 µm. c NETs were quantified using Fiji and expressed as percentage of NETs, percentage of staining under NETs per field of view and total area under NET staining in square millimeters. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, centre line and columns indicate mean values ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 and error bars denote standard deviations
    Figure Legend Snippet: DNase facilitates bacterial clearance in a rat model of pneumococcal meningitis. Rats received a subarachnoid infusion of either S. pneumoniae SP001 strain (infected) or equal volume of saline solution (control). a After the rats were sacrificed, the brain, the right lung, and the spleen were collected and homogenized immediately. The blood was collected and centrifuged to obtain plasma. Organ homogenates and blood plasma samples were spread onto agar plates and resulting bacterial colonies were counted after 24 h. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and error bars denote standard deviations. b To determine the effect of intrathecal DNase treatment, infected rats either received a subarachnoid infusion of 10 units of DNase simultaneously (0 h) or 10 h after the infection, or they received an equal volume of saline vehicle solution simultaneously to the infection. To determine the effect of intravenous DNase treatment, infected rats either received an intravenous bolus dose of 3500 units of DNase 6 h after the infection, followed by intravenous infusion of 780 units/h over the next 18 h, or they received an equal volume of saline vehicle control in the same manner. In all cases, uninfected (control) rats received an equal volume of saline vehicle control either intrathecally or intravenously as indicated. All rats were sacrificed 24 h after the infection. Cerebrospinal fluid was collected for visualization of NETs only by immunofluorescence against rat myeloperoxidase (red) and DNA (blue). Areas of red and blue colocalization represent NETs. Scale bars denote 200 µm. c NETs were quantified using Fiji and expressed as percentage of NETs, percentage of staining under NETs per field of view and total area under NET staining in square millimeters. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, centre line and columns indicate mean values ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 and error bars denote standard deviations

    Techniques Used: Infection, Immunofluorescence, Staining

    3) Product Images from "Initiation-specific alleles of the Cdc45 helicase-activating protein"

    Article Title: Initiation-specific alleles of the Cdc45 helicase-activating protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0214426

    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Tube Formation Assay

    Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Produced, Agarose Gel Electrophoresis, Mutagenesis

    4) Product Images from "Microfabricated Engineered Particle Systems for Respiratory Drug Delivery and Other Pharmaceutical Applications"

    Article Title: Microfabricated Engineered Particle Systems for Respiratory Drug Delivery and Other Pharmaceutical Applications

    Journal: Journal of Drug Delivery

    doi: 10.1155/2012/941243

    SEM micrographs of diverse PRINT aerosols. (a) BSA/Lactose 200 × 200 nm cylinders; (b) IgG/Lactose10 μ m pollen; (c) 30 K PLGA 3 μ m cylinders; (d) itraconazole 1.5 μ m torus; (e) itraconazole 3 μ m torus; (f) itraconazole 6 μ m torus; (g) zanamivir 1.5 μ m torus; (h) DNAse 1.5 μ m torus; (i) siRNA 1.5 μ m torus.
    Figure Legend Snippet: SEM micrographs of diverse PRINT aerosols. (a) BSA/Lactose 200 × 200 nm cylinders; (b) IgG/Lactose10 μ m pollen; (c) 30 K PLGA 3 μ m cylinders; (d) itraconazole 1.5 μ m torus; (e) itraconazole 3 μ m torus; (f) itraconazole 6 μ m torus; (g) zanamivir 1.5 μ m torus; (h) DNAse 1.5 μ m torus; (i) siRNA 1.5 μ m torus.

    Techniques Used:

    Related Articles

    Isolation:

    Article Title: BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking CLU+ CAF expression with HSF1 signaling
    Article Snippet: .. Primary PSC isolation: pancreata were collected postmortem from Hsf1 null mice or WT littermates into HBSS (Sigma-Aldrich, H6648), then minced into Roswell Park Memorial Institute 1640 (RPMI) (Biological industries, 01-100-1A), supplemented with 0.5mg/mL Collagenase D (Merck, 11088866001), 0.1mg/mL Deoxyribonuclease I (Worthington, LS002007) and 1mM HEPES (Biological Industries, 03-025-1B). ..

    Article Title: The oxidation‐resistant CaMKII‐MM281/282VV mutation does not prevent arrhythmias in CPVT1). The oxidation‐resistant CaMKII‐MM281/282VV mutation does not prevent arrhythmias in CPVT1
    Article Snippet: .. Following digestion for 6–10 min, the left ventricle was isolated, transferred into isolation buffer with 0.02 mM Ca2+ and 0.1% bovine serum albumin, cut into pieces, and triturated with ~100 U/mL of deoxyribonuclease I (Worthington). ..

    Mouse Assay:

    Article Title: BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking CLU+ CAF expression with HSF1 signaling
    Article Snippet: .. Primary PSC isolation: pancreata were collected postmortem from Hsf1 null mice or WT littermates into HBSS (Sigma-Aldrich, H6648), then minced into Roswell Park Memorial Institute 1640 (RPMI) (Biological industries, 01-100-1A), supplemented with 0.5mg/mL Collagenase D (Merck, 11088866001), 0.1mg/mL Deoxyribonuclease I (Worthington, LS002007) and 1mM HEPES (Biological Industries, 03-025-1B). ..

    Lysis:

    Article Title: The periphilin 1-like BFAR isoform 3 is highly expressed in transcriptionally silent oocytes and involved in RNA metabolism
    Article Snippet: .. After lysis of nuclear pellets, extracts were additionally supplemented with MgCl2 to 1.5 mM and digested with DNaseI (5 U; Worthington Biochemical Corporation) for 30 min at room temperature. ..

    Blocking Assay:

    Article Title: Three-axis classification of mouse lung mesenchymal cells reveals two populations of myofibroblasts
    Article Snippet: .. Lung lobes were minced with forceps and digested with 2 mg/mL Collagenase Type I (Worthington, CLS-1, LS004197), 2 mg/mL Elastase (Worthington, ESL, LS002294), and 0.5 mg/mL DNase I (Worthington, D, LS002007) for 30 min at 37°C using a dry block incubator. ..

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  • 95
    Worthington Biochemical dnase i
    Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c)  “Run-on” activity assayed later during fractionation (as in  a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by
    Dnase I, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Worthington Biochemical
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2021-10
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    Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c)  “Run-on” activity assayed later during fractionation (as in  a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

    Journal: Nature methods

    Article Title: The proteomes of transcription factories containing RNA polymerases I, II or III

    doi: 10.1038/nmeth.1705

    Figure Lengend Snippet: Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c) “Run-on” activity assayed later during fractionation (as in a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

    Article Snippet: Resuspended nuclei were digested (30 min; 33°C) with either (i) DNase I (protease- and RNase-free; Worthington;10 units/107 cells in 100 μl PB plus 0.5 mM CaCl2 ), or (ii) Hae III (Invitrogen; 1000 units/107 cells), or (iii) Hind III (New England Biolabs; 1000 units/107 cells) in PB; reactions were stopped by adding EDTA to 2.5 mM and cooling in iced water.

    Techniques: Purification, Mass Spectrometry, Activity Assay, Fractionation

    Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the  CD4  gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.

    Journal: PLoS ONE

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    doi: 10.1371/journal.pone.0085740

    Figure Lengend Snippet: Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the CD4 gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.

    Article Snippet: To increase the reproducibility and minimize experimental variations, we chose to digest the nuclei of CD4 T cells with various amounts of DNase I on ice for a relatively long period of time (1 hr).

    Techniques: Polymerase Chain Reaction, Magnetic Beads, Amplification, Real-time Polymerase Chain Reaction, Isolation

    Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.

    Journal: PLoS ONE

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    doi: 10.1371/journal.pone.0085740

    Figure Lengend Snippet: Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.

    Article Snippet: To increase the reproducibility and minimize experimental variations, we chose to digest the nuclei of CD4 T cells with various amounts of DNase I on ice for a relatively long period of time (1 hr).

    Techniques: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Molecular Weight, Derivative Assay, Amplification

    Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.

    Journal: PLoS ONE

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    doi: 10.1371/journal.pone.0085740

    Figure Lengend Snippet: Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.

    Article Snippet: To increase the reproducibility and minimize experimental variations, we chose to digest the nuclei of CD4 T cells with various amounts of DNase I on ice for a relatively long period of time (1 hr).

    Techniques: Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Isolation, Magnetic Beads, Amplification

    DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.

    Journal: PLoS ONE

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    doi: 10.1371/journal.pone.0085740

    Figure Lengend Snippet: DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.

    Article Snippet: To increase the reproducibility and minimize experimental variations, we chose to digest the nuclei of CD4 T cells with various amounts of DNase I on ice for a relatively long period of time (1 hr).

    Techniques: Isolation, Agarose Gel Electrophoresis, Purification, FACS