pbr322 dna msp  (New England Biolabs)


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    New England Biolabs pbr322 dna msp
    Cells with fast initial decay kinetics express β 1 subunit mRNA A and B , scatter plots of IPSC 90%-width versus peak current amplitude for two RTN cells recorded on the same day. Each point in A and B represents an individual IPSC. A is a recording from a cell dominated with slow and small IPSCs (○) and B , represents a recording dominated by brief and large IPSCs ( ). C , ensemble averaged IPSCs averaged from isolated spontaneous inhibitory events (selected on the basis of having arisen from an event-free baseline and showing complete decay to the same baseline) for the two cells represented in A (black, n = 258) and B (grey, n = 217). Each trace was best fitted to two exponentials (dotted line superimposed on each trace). Note: the cell dominated with fast IPSCs yielded a ensemble averaged IPSC with a larger A 1 value than A 2 whereas the opposite is true for the slower cell. D , ethidium bromide stained 8% polyacrylamide gel showing single-cell RT-PCR analysis from five RTN cells examined in one day of recording, two of the cells are represented in A (lane 3) and B (lane 7). Cells positive for the β 1 subunit cDNA showed a solitary band at 162 bp after two rounds of amplification using nested primers (lanes 6 and 7, see methods). Lanes 1 and 2 are water and media controls that underwent reverse transcription along with individual cells in lanes 3 through 7; lane 8 is a positive control using 40 pg of total RNA; lanes 9 and 10 are water controls used in the master mix for each round of PCR amplification. The molecular weight marker in lane 11 is the <t>Msp</t> I digest of <t>pBR322.</t> The size of each fragment is noted on the right. The parameters for β 1 negative cells in lane 4: A 1 = 9.80, τ 1 = 13.75, A 2 = 14.17, τ 2 = 64.61, τ DW = 43.82l; and lane 5: A 1 = 12.94, τ 1 = 12.94, A 2 = 5.69, τ 2 = 77.21, τ DW = 29.63. The β 1 positive cell in lane 6: A 1 = 18.05, τ 1 = 15.11, A 2 = 4.15, τ 2 = 74.44, τ DW = 26.20.
    Pbr322 Dna Msp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Fast IPSCs in rat thalamic reticular nucleus require the GABAA receptor ?1 subunit"

    Article Title: Fast IPSCs in rat thalamic reticular nucleus require the GABAA receptor ?1 subunit

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2006.106617

    Cells with fast initial decay kinetics express β 1 subunit mRNA A and B , scatter plots of IPSC 90%-width versus peak current amplitude for two RTN cells recorded on the same day. Each point in A and B represents an individual IPSC. A is a recording from a cell dominated with slow and small IPSCs (○) and B , represents a recording dominated by brief and large IPSCs ( ). C , ensemble averaged IPSCs averaged from isolated spontaneous inhibitory events (selected on the basis of having arisen from an event-free baseline and showing complete decay to the same baseline) for the two cells represented in A (black, n = 258) and B (grey, n = 217). Each trace was best fitted to two exponentials (dotted line superimposed on each trace). Note: the cell dominated with fast IPSCs yielded a ensemble averaged IPSC with a larger A 1 value than A 2 whereas the opposite is true for the slower cell. D , ethidium bromide stained 8% polyacrylamide gel showing single-cell RT-PCR analysis from five RTN cells examined in one day of recording, two of the cells are represented in A (lane 3) and B (lane 7). Cells positive for the β 1 subunit cDNA showed a solitary band at 162 bp after two rounds of amplification using nested primers (lanes 6 and 7, see methods). Lanes 1 and 2 are water and media controls that underwent reverse transcription along with individual cells in lanes 3 through 7; lane 8 is a positive control using 40 pg of total RNA; lanes 9 and 10 are water controls used in the master mix for each round of PCR amplification. The molecular weight marker in lane 11 is the Msp I digest of pBR322. The size of each fragment is noted on the right. The parameters for β 1 negative cells in lane 4: A 1 = 9.80, τ 1 = 13.75, A 2 = 14.17, τ 2 = 64.61, τ DW = 43.82l; and lane 5: A 1 = 12.94, τ 1 = 12.94, A 2 = 5.69, τ 2 = 77.21, τ DW = 29.63. The β 1 positive cell in lane 6: A 1 = 18.05, τ 1 = 15.11, A 2 = 4.15, τ 2 = 74.44, τ DW = 26.20.
    Figure Legend Snippet: Cells with fast initial decay kinetics express β 1 subunit mRNA A and B , scatter plots of IPSC 90%-width versus peak current amplitude for two RTN cells recorded on the same day. Each point in A and B represents an individual IPSC. A is a recording from a cell dominated with slow and small IPSCs (○) and B , represents a recording dominated by brief and large IPSCs ( ). C , ensemble averaged IPSCs averaged from isolated spontaneous inhibitory events (selected on the basis of having arisen from an event-free baseline and showing complete decay to the same baseline) for the two cells represented in A (black, n = 258) and B (grey, n = 217). Each trace was best fitted to two exponentials (dotted line superimposed on each trace). Note: the cell dominated with fast IPSCs yielded a ensemble averaged IPSC with a larger A 1 value than A 2 whereas the opposite is true for the slower cell. D , ethidium bromide stained 8% polyacrylamide gel showing single-cell RT-PCR analysis from five RTN cells examined in one day of recording, two of the cells are represented in A (lane 3) and B (lane 7). Cells positive for the β 1 subunit cDNA showed a solitary band at 162 bp after two rounds of amplification using nested primers (lanes 6 and 7, see methods). Lanes 1 and 2 are water and media controls that underwent reverse transcription along with individual cells in lanes 3 through 7; lane 8 is a positive control using 40 pg of total RNA; lanes 9 and 10 are water controls used in the master mix for each round of PCR amplification. The molecular weight marker in lane 11 is the Msp I digest of pBR322. The size of each fragment is noted on the right. The parameters for β 1 negative cells in lane 4: A 1 = 9.80, τ 1 = 13.75, A 2 = 14.17, τ 2 = 64.61, τ DW = 43.82l; and lane 5: A 1 = 12.94, τ 1 = 12.94, A 2 = 5.69, τ 2 = 77.21, τ DW = 29.63. The β 1 positive cell in lane 6: A 1 = 18.05, τ 1 = 15.11, A 2 = 4.15, τ 2 = 74.44, τ DW = 26.20.

    Techniques Used: Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Polymerase Chain Reaction, Molecular Weight, Marker

    2) Product Images from "Time-Resolved Analysis of Transcription through Chromatin"

    Article Title: Time-Resolved Analysis of Transcription through Chromatin

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2019.01.016

    Time course of in vitro Pol II transcription through a nucleosome in the absence/presence of human FACT ], with permission). The in vitro Pol II transcription through the 603 nucleosome in the absence or presence of hFACT was conducted at 150 mM KCl for indicated time intervals (0, 5, 10, 15, 20, 25, 30, 60 seconds). After reactions, the pulse-labeled RNA was purified and analyzed by denaturing PAGE. The image was then obtained using phosphoimager and analyzed using OptiQuant software. The position of the nucleosome on the DNA template is indicated. The bands corresponding to nucleosome-specific pausing sites are quantified. The quantified bands are then grouped into 10 clusters of the pausing intermediates (A to J) and the run-off (K). M: pBR322 DNA- Msp I digest.
    Figure Legend Snippet: Time course of in vitro Pol II transcription through a nucleosome in the absence/presence of human FACT ], with permission). The in vitro Pol II transcription through the 603 nucleosome in the absence or presence of hFACT was conducted at 150 mM KCl for indicated time intervals (0, 5, 10, 15, 20, 25, 30, 60 seconds). After reactions, the pulse-labeled RNA was purified and analyzed by denaturing PAGE. The image was then obtained using phosphoimager and analyzed using OptiQuant software. The position of the nucleosome on the DNA template is indicated. The bands corresponding to nucleosome-specific pausing sites are quantified. The quantified bands are then grouped into 10 clusters of the pausing intermediates (A to J) and the run-off (K). M: pBR322 DNA- Msp I digest.

    Techniques Used: In Vitro, Labeling, Purification, Polyacrylamide Gel Electrophoresis, Software

    3) Product Images from "SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12 *"

    Article Title: SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.266619

    Competition between H-NS and SlyA binding upstream of the fimB promoter. A , binding of H-NS to the fim03 fragment labeled at the fim03f end. Decreasing concentrations of H-NS were mixed with the fim03 DNA with and without SlyA in the “HNS” buffer. Lane 1 , no proteins; lanes 2 and 7 , 400 n m H-NS; lanes 3 and 8 , 200 n m H-NS; lanes 4 and 9 , 100 n m H-NS; lanes 5 and 10 , 50 n m H-NS; lanes 6 and 12 , 25 n m H-NS; lanes 7–12 also contained 2 μ m SlyA. B , H-NS binding with and without SlyA in the “SlyA” buffer. Lane 1 , no proteins; lane 2 , 50 n m SlyA; lane 3 , 1 μ m SlyA; lane 4 , 50 n m H-NS; lane 5 , 100 n m H-NS; lane 6 , 1 μ m SlyA, and 50 n m H-NS; lane 7, 1 μ m SlyA and 100 n m H-NS. Proteins were incubated for 15 min at 25 °C before treatment with DNase I. Products were analyzed on 6% denaturing polyacrylamide gels. Regions protected by H-NS and SlyA are indicated. The arrows indicate hypersensitive DNaseI cleavages on the DNA in the presence of H-NS, which are observed in the “SlyA” buffer ( B ) but not in the “H-NS” buffer (H-NS). The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).
    Figure Legend Snippet: Competition between H-NS and SlyA binding upstream of the fimB promoter. A , binding of H-NS to the fim03 fragment labeled at the fim03f end. Decreasing concentrations of H-NS were mixed with the fim03 DNA with and without SlyA in the “HNS” buffer. Lane 1 , no proteins; lanes 2 and 7 , 400 n m H-NS; lanes 3 and 8 , 200 n m H-NS; lanes 4 and 9 , 100 n m H-NS; lanes 5 and 10 , 50 n m H-NS; lanes 6 and 12 , 25 n m H-NS; lanes 7–12 also contained 2 μ m SlyA. B , H-NS binding with and without SlyA in the “SlyA” buffer. Lane 1 , no proteins; lane 2 , 50 n m SlyA; lane 3 , 1 μ m SlyA; lane 4 , 50 n m H-NS; lane 5 , 100 n m H-NS; lane 6 , 1 μ m SlyA, and 50 n m H-NS; lane 7, 1 μ m SlyA and 100 n m H-NS. Proteins were incubated for 15 min at 25 °C before treatment with DNase I. Products were analyzed on 6% denaturing polyacrylamide gels. Regions protected by H-NS and SlyA are indicated. The arrows indicate hypersensitive DNaseI cleavages on the DNA in the presence of H-NS, which are observed in the “SlyA” buffer ( B ) but not in the “H-NS” buffer (H-NS). The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Techniques Used: Binding Assay, Labeling, Incubation, Marker

    The interaction of SlyA with the fimB promoter region in vitro . A , effect of 0, 15, 30, 60, and 120 n m SlyA dimer on the electophoretic mobility of DNA amplicons fim03 and pBS (each 11 n m ). Amplicon fim03 is 282 bp of the region upstream of fimB and includes O SA1 and O SA2 ( Fig. 1 ). Amplicon pBS is a negative control as previously described ( 27 ). The SlyA-DNA complex is indicated with an arrow . Samples were separated on a 5% polyacrylamide gel. Electrophoresis was carried out at 160 V for 35 min as described under “Experimental Procedures.” B , DNase I footprinting. The fim03 fragment, labeled at the fim03r end, was mixed with decreasing concentrations of SlyA for 15 min at 25 °C before digestion with DNaseI. Lane 1 , no SlyA; lane 2 , 1 μ m SlyA; lane 3 , 500 n m ; lane 4 , 250 n m ; lane 5 , 125 n m ; lane 6 , 62.5 n m ; lane 7 , 31 n m ; lane 8 , 15.6 n m . The products were analyzed on a 6% denaturing polyacrylamide gel. Regions protected by SlyA are indicated. The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).
    Figure Legend Snippet: The interaction of SlyA with the fimB promoter region in vitro . A , effect of 0, 15, 30, 60, and 120 n m SlyA dimer on the electophoretic mobility of DNA amplicons fim03 and pBS (each 11 n m ). Amplicon fim03 is 282 bp of the region upstream of fimB and includes O SA1 and O SA2 ( Fig. 1 ). Amplicon pBS is a negative control as previously described ( 27 ). The SlyA-DNA complex is indicated with an arrow . Samples were separated on a 5% polyacrylamide gel. Electrophoresis was carried out at 160 V for 35 min as described under “Experimental Procedures.” B , DNase I footprinting. The fim03 fragment, labeled at the fim03r end, was mixed with decreasing concentrations of SlyA for 15 min at 25 °C before digestion with DNaseI. Lane 1 , no SlyA; lane 2 , 1 μ m SlyA; lane 3 , 500 n m ; lane 4 , 250 n m ; lane 5 , 125 n m ; lane 6 , 62.5 n m ; lane 7 , 31 n m ; lane 8 , 15.6 n m . The products were analyzed on a 6% denaturing polyacrylamide gel. Regions protected by SlyA are indicated. The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Techniques Used: In Vitro, Amplification, Negative Control, Polyacrylamide Gel Electrophoresis, Footprinting, Labeling, Marker

    4) Product Images from "Comparison of Cytotoxic, Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells"

    Article Title: Comparison of Cytotoxic, Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23105339

    DNA-protective activity of skyrin (SK) analyzed by DNA-topology assay. Electrophoretic monitoring of changes induced by FeSO 4 × 7H 2 O in the topology of plasmid DNA (pBR322) after skyrin application (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + FeSO 4 × 7H 2 O + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.
    Figure Legend Snippet: DNA-protective activity of skyrin (SK) analyzed by DNA-topology assay. Electrophoretic monitoring of changes induced by FeSO 4 × 7H 2 O in the topology of plasmid DNA (pBR322) after skyrin application (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + FeSO 4 × 7H 2 O + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.

    Techniques Used: Activity Assay, Plasmid Preparation, Negative Control, Positive Control

    Potential DNA-damaging effects of skyrin on plasmid DNA. Electrophoretic monitoring of changes in the topology of plasmid DNA (pBR322) induced by skyrin (SK) using DNA-topology assay (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.
    Figure Legend Snippet: Potential DNA-damaging effects of skyrin on plasmid DNA. Electrophoretic monitoring of changes in the topology of plasmid DNA (pBR322) induced by skyrin (SK) using DNA-topology assay (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.

    Techniques Used: Plasmid Preparation, Negative Control, Positive Control

    Evaluation of the potential DNA-protective effects of skyrin (SK) on the HepG2 cell line (24 h treatment) at different concentrations using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 1 mM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (24 h treatment), and hydrogen peroxide (1 mM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p
    Figure Legend Snippet: Evaluation of the potential DNA-protective effects of skyrin (SK) on the HepG2 cell line (24 h treatment) at different concentrations using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 1 mM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (24 h treatment), and hydrogen peroxide (1 mM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p

    Techniques Used: Single Cell Gel Electrophoresis, Negative Control, Positive Control, Concentration Assay

    Evaluation of the potential DNA-protective effects of skyrin (SK) on human lymphocytes (1 h treatment) at different concentrations, using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 440 μM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (1 h treatment), and hydrogen peroxide (440 μM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p
    Figure Legend Snippet: Evaluation of the potential DNA-protective effects of skyrin (SK) on human lymphocytes (1 h treatment) at different concentrations, using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 440 μM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (1 h treatment), and hydrogen peroxide (440 μM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p

    Techniques Used: Single Cell Gel Electrophoresis, Negative Control, Positive Control, Concentration Assay

    5) Product Images from "Cylindrical Illumination Confocal Spectroscopy: Rectifying the Limitations of Confocal Single Molecule Spectroscopy through One-Dimensional Beam Shaping"

    Article Title: Cylindrical Illumination Confocal Spectroscopy: Rectifying the Limitations of Confocal Single Molecule Spectroscopy through One-Dimensional Beam Shaping

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.108.132472

    Burst size distribution analysis histograms of PicoGreen-stained pBR322 DNA taken using standard SMD ( left ) and CICS ( right ). In standard SMD, the DNA peak is not resolved from the noise fluctuations due to the Gaussian OV profile whereas CICS shows a clearly discernible peak due to the high uniformity of the OV profile.
    Figure Legend Snippet: Burst size distribution analysis histograms of PicoGreen-stained pBR322 DNA taken using standard SMD ( left ) and CICS ( right ). In standard SMD, the DNA peak is not resolved from the noise fluctuations due to the Gaussian OV profile whereas CICS shows a clearly discernible peak due to the high uniformity of the OV profile.

    Techniques Used: Staining

    Experimental single molecule trace data of TOTO-3 stained pBR322 DNA taken using SMD ( top ) and CICS ( bottom ). The CICS experimental data shows a high burst rate and burst height uniformity that parallels the results of the Monte Carlo simulations. The bin time was 0.1 ms.
    Figure Legend Snippet: Experimental single molecule trace data of TOTO-3 stained pBR322 DNA taken using SMD ( top ) and CICS ( bottom ). The CICS experimental data shows a high burst rate and burst height uniformity that parallels the results of the Monte Carlo simulations. The bin time was 0.1 ms.

    Techniques Used: Staining, Mass Spectrometry

    6) Product Images from "ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile"

    Article Title: ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

    Journal: Molecular Biology International

    doi: 10.1155/2012/283974

    ASGR1 transcript profiles in different individuals. Nested PCR was used to detect ASGR1 transcripts T1 and T2 (first round primers A1CDSF1 + A1CDSR1; second round A1TEST2 + ASG1RTR). A product of 408 bp represented T1, present in RNA from the M + L cell fraction of five individuals (A–E) and also in HepG2 and THP1; a 291 bp product indicated the presence of T2 (individual E, and, very faintly, in HepG2). The RT-PCR product for individual A was coloaded with the DNA size standard (pBR322/ Msp I, 238 bp and 120 bp sizes indicated). Analysis was done using agarose gel electrophoresis. The figure is a composite of various data (indicated by individual gel windows).
    Figure Legend Snippet: ASGR1 transcript profiles in different individuals. Nested PCR was used to detect ASGR1 transcripts T1 and T2 (first round primers A1CDSF1 + A1CDSR1; second round A1TEST2 + ASG1RTR). A product of 408 bp represented T1, present in RNA from the M + L cell fraction of five individuals (A–E) and also in HepG2 and THP1; a 291 bp product indicated the presence of T2 (individual E, and, very faintly, in HepG2). The RT-PCR product for individual A was coloaded with the DNA size standard (pBR322/ Msp I, 238 bp and 120 bp sizes indicated). Analysis was done using agarose gel electrophoresis. The figure is a composite of various data (indicated by individual gel windows).

    Techniques Used: Nested PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    ASGR2 transcript profiles in different individuals. Primer pair ASGR2RTF3 + ASGR2RTR2 gave RT-PCR products of different length (arrowed) according to the ASGR2 transcripts present. RNA extracted from the M + L cell fraction of 6 different individuals (A–F) gave either a profile consistent with transcripts encoding all isoforms (a, b, c, and d) or just with isoforms b and d. The liver cell line HepG2 showed all transcripts, the monocyte cell line THP1 showed only transcripts encoding isoforms b and d. The analysis was done using polyacrylamide gel electrophoresis. DNA size reference was pBR322/ Msp I (“Marker”, 238 bp and 120 bp sizes indicated). In addition to the expected products, additional major bands were visualized on polyacrylamide gel electrophoresis of the RT-PCR screen for ASGR2 transcripts (bracketed bands). It was considered possible that these represented heteroduplexes arising by cross-hybridization of complementary strands of the closely homologous true PCR products. To test this, each candidate heteroduplex band was excised from the gel, eluted into water, reamplified using the primers used in the initial PCR and the products electrophoresed on polyacrylamide. The results confirmed that the additional bands were hybrid duplexes containing complementary strands from nonidentical true PCR products: each hybrid yielded products consistent with amplification of two different template strands (data not shown).
    Figure Legend Snippet: ASGR2 transcript profiles in different individuals. Primer pair ASGR2RTF3 + ASGR2RTR2 gave RT-PCR products of different length (arrowed) according to the ASGR2 transcripts present. RNA extracted from the M + L cell fraction of 6 different individuals (A–F) gave either a profile consistent with transcripts encoding all isoforms (a, b, c, and d) or just with isoforms b and d. The liver cell line HepG2 showed all transcripts, the monocyte cell line THP1 showed only transcripts encoding isoforms b and d. The analysis was done using polyacrylamide gel electrophoresis. DNA size reference was pBR322/ Msp I (“Marker”, 238 bp and 120 bp sizes indicated). In addition to the expected products, additional major bands were visualized on polyacrylamide gel electrophoresis of the RT-PCR screen for ASGR2 transcripts (bracketed bands). It was considered possible that these represented heteroduplexes arising by cross-hybridization of complementary strands of the closely homologous true PCR products. To test this, each candidate heteroduplex band was excised from the gel, eluted into water, reamplified using the primers used in the initial PCR and the products electrophoresed on polyacrylamide. The results confirmed that the additional bands were hybrid duplexes containing complementary strands from nonidentical true PCR products: each hybrid yielded products consistent with amplification of two different template strands (data not shown).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Hybridization, Polymerase Chain Reaction, Amplification

    Real-time PCR analyses. (a) Agarose gel electrophoresis of products from optimized real-time PCRs for ASGR1 (113 bp, primers ASG1RTF + ASG1RTR), ASGR2 (171 bp, primers ASG2RTF + ASG2RTR), and B2M (231 bp, primers B2MRTF + B2MRTR). Substrate was HepG2 cDNA. (b) Melting curve analysis of real-time PCR products from panel (a). The Tm for each product is indicated. (c and d) Relative quantification of ASGR1 (c) and ASGR2 (d) transcripts in cell-sorted monocytes (MC) compared with HepG2 (G2). B2M was used as the reference. (i) Fluorescence profiles obtained during real-time PCR. Samples were analyzed in duplicate, for clarity only one of each duplicate is shown. (ii) Melting curve analysis for the real-time PCR products in (i). (iii) Agarose gel electrophoresis of real-time PCR products (duplicate analyses shown). Neg indicates real-time PCR control containing water instead of nucleic acid template. In panels a, c(iii), and d(iii), M denotes pBR322/ Msp I size standard (238 bp and 120 bp sizes indicated).
    Figure Legend Snippet: Real-time PCR analyses. (a) Agarose gel electrophoresis of products from optimized real-time PCRs for ASGR1 (113 bp, primers ASG1RTF + ASG1RTR), ASGR2 (171 bp, primers ASG2RTF + ASG2RTR), and B2M (231 bp, primers B2MRTF + B2MRTR). Substrate was HepG2 cDNA. (b) Melting curve analysis of real-time PCR products from panel (a). The Tm for each product is indicated. (c and d) Relative quantification of ASGR1 (c) and ASGR2 (d) transcripts in cell-sorted monocytes (MC) compared with HepG2 (G2). B2M was used as the reference. (i) Fluorescence profiles obtained during real-time PCR. Samples were analyzed in duplicate, for clarity only one of each duplicate is shown. (ii) Melting curve analysis for the real-time PCR products in (i). (iii) Agarose gel electrophoresis of real-time PCR products (duplicate analyses shown). Neg indicates real-time PCR control containing water instead of nucleic acid template. In panels a, c(iii), and d(iii), M denotes pBR322/ Msp I size standard (238 bp and 120 bp sizes indicated).

    Techniques Used: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Fluorescence

    Detection of ASGR1 and ASGR2 transcripts in peripheral blood mononuclear cells and localization to monocytes. Transcripts were detected using RT-PCR, the presence of ASGR1 transcripts was indicated by a 113 bp amplification product (primers ASG1RTF + ASG1RTR) and ASGR2 transcripts by a 171 bp product (primers ASG2RTF + ASG2RTR). HepG2 was used as a positive control and water as a negative control. Where necessary, β -2-microglobulin ( B2M ) was used as a reference to demonstrate equivalent amplification for each RNA preparation, (primers B2MRTF + B2MRTR, product 231 bp). (a) Analysis of RNA from monocyte cell line THP1 and PBMCs from two unrelated individuals (1 and 2). ASGR1 and ASGR2 transcripts were detected in all cases. (b) Analysis of RNA from peripheral blood cell fractions: monocytes + lymphocytes (M + L) and granulocytes + lymphocytes (G + L). ASGR1 and ASGR2 transcripts were detected in M + L but not in G + L. (c) Analysis of RNA from cell-sorted monocytes (mono, lanes 1–3 triplicate analyses) and lymphocytes (lymph, lanes 1–3 triplicate analyses) (data for water control not shown for ASGR1 and ASGR2 ). The data were obtained from a single experiment involving several gels: these are indicated by separate gel windows. For each panel, analysis was done using agarose gel electrophoresis with pBR322/ Msp I size standard (“Marker”, 238 bp and 120 bp indicated).
    Figure Legend Snippet: Detection of ASGR1 and ASGR2 transcripts in peripheral blood mononuclear cells and localization to monocytes. Transcripts were detected using RT-PCR, the presence of ASGR1 transcripts was indicated by a 113 bp amplification product (primers ASG1RTF + ASG1RTR) and ASGR2 transcripts by a 171 bp product (primers ASG2RTF + ASG2RTR). HepG2 was used as a positive control and water as a negative control. Where necessary, β -2-microglobulin ( B2M ) was used as a reference to demonstrate equivalent amplification for each RNA preparation, (primers B2MRTF + B2MRTR, product 231 bp). (a) Analysis of RNA from monocyte cell line THP1 and PBMCs from two unrelated individuals (1 and 2). ASGR1 and ASGR2 transcripts were detected in all cases. (b) Analysis of RNA from peripheral blood cell fractions: monocytes + lymphocytes (M + L) and granulocytes + lymphocytes (G + L). ASGR1 and ASGR2 transcripts were detected in M + L but not in G + L. (c) Analysis of RNA from cell-sorted monocytes (mono, lanes 1–3 triplicate analyses) and lymphocytes (lymph, lanes 1–3 triplicate analyses) (data for water control not shown for ASGR1 and ASGR2 ). The data were obtained from a single experiment involving several gels: these are indicated by separate gel windows. For each panel, analysis was done using agarose gel electrophoresis with pBR322/ Msp I size standard (“Marker”, 238 bp and 120 bp indicated).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Negative Control, Agarose Gel Electrophoresis

    7) Product Images from "Cylindrical Illumination Confocal Spectroscopy: Rectifying the Limitations of Confocal Single Molecule Spectroscopy through One-Dimensional Beam Shaping"

    Article Title: Cylindrical Illumination Confocal Spectroscopy: Rectifying the Limitations of Confocal Single Molecule Spectroscopy through One-Dimensional Beam Shaping

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.108.132472

    Burst size distribution analysis histograms of PicoGreen-stained pBR322 DNA taken using standard SMD ( left ) and CICS ( right ). In standard SMD, the DNA peak is not resolved from the noise fluctuations due to the Gaussian OV profile whereas CICS shows a clearly discernible peak due to the high uniformity of the OV profile.
    Figure Legend Snippet: Burst size distribution analysis histograms of PicoGreen-stained pBR322 DNA taken using standard SMD ( left ) and CICS ( right ). In standard SMD, the DNA peak is not resolved from the noise fluctuations due to the Gaussian OV profile whereas CICS shows a clearly discernible peak due to the high uniformity of the OV profile.

    Techniques Used: Staining

    Experimental single molecule trace data of TOTO-3 stained pBR322 DNA taken using SMD ( top ) and CICS ( bottom ). The CICS experimental data shows a high burst rate and burst height uniformity that parallels the results of the Monte Carlo simulations. The bin time was 0.1 ms.
    Figure Legend Snippet: Experimental single molecule trace data of TOTO-3 stained pBR322 DNA taken using SMD ( top ) and CICS ( bottom ). The CICS experimental data shows a high burst rate and burst height uniformity that parallels the results of the Monte Carlo simulations. The bin time was 0.1 ms.

    Techniques Used: Staining, Mass Spectrometry

    8) Product Images from "Cylindrical Illumination Confocal Spectroscopy: Rectifying the Limitations of Confocal Single Molecule Spectroscopy through One-Dimensional Beam Shaping"

    Article Title: Cylindrical Illumination Confocal Spectroscopy: Rectifying the Limitations of Confocal Single Molecule Spectroscopy through One-Dimensional Beam Shaping

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.108.132472

    Burst size distribution analysis histograms of PicoGreen-stained pBR322 DNA taken using standard SMD ( left ) and CICS ( right ). In standard SMD, the DNA peak is not resolved from the noise fluctuations due to the Gaussian OV profile whereas CICS shows a clearly discernible peak due to the high uniformity of the OV profile.
    Figure Legend Snippet: Burst size distribution analysis histograms of PicoGreen-stained pBR322 DNA taken using standard SMD ( left ) and CICS ( right ). In standard SMD, the DNA peak is not resolved from the noise fluctuations due to the Gaussian OV profile whereas CICS shows a clearly discernible peak due to the high uniformity of the OV profile.

    Techniques Used: Staining

    Experimental single molecule trace data of TOTO-3 stained pBR322 DNA taken using SMD ( top ) and CICS ( bottom ). The CICS experimental data shows a high burst rate and burst height uniformity that parallels the results of the Monte Carlo simulations. The bin time was 0.1 ms.
    Figure Legend Snippet: Experimental single molecule trace data of TOTO-3 stained pBR322 DNA taken using SMD ( top ) and CICS ( bottom ). The CICS experimental data shows a high burst rate and burst height uniformity that parallels the results of the Monte Carlo simulations. The bin time was 0.1 ms.

    Techniques Used: Staining, Mass Spectrometry

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    • pbr322 dna msp  (New England Biolabs)
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    New England Biolabs pbr322 dna msp
    Cells with fast initial decay kinetics express β 1 subunit mRNA A and B , scatter plots of IPSC 90%-width versus peak current amplitude for two RTN cells recorded on the same day. Each point in A and B represents an individual IPSC. A is a recording from a cell dominated with slow and small IPSCs (○) and B , represents a recording dominated by brief and large IPSCs ( ). C , ensemble averaged IPSCs averaged from isolated spontaneous inhibitory events (selected on the basis of having arisen from an event-free baseline and showing complete decay to the same baseline) for the two cells represented in A (black, n = 258) and B (grey, n = 217). Each trace was best fitted to two exponentials (dotted line superimposed on each trace). Note: the cell dominated with fast IPSCs yielded a ensemble averaged IPSC with a larger A 1 value than A 2 whereas the opposite is true for the slower cell. D , ethidium bromide stained 8% polyacrylamide gel showing single-cell RT-PCR analysis from five RTN cells examined in one day of recording, two of the cells are represented in A (lane 3) and B (lane 7). Cells positive for the β 1 subunit cDNA showed a solitary band at 162 bp after two rounds of amplification using nested primers (lanes 6 and 7, see methods). Lanes 1 and 2 are water and media controls that underwent reverse transcription along with individual cells in lanes 3 through 7; lane 8 is a positive control using 40 pg of total RNA; lanes 9 and 10 are water controls used in the master mix for each round of PCR amplification. The molecular weight marker in lane 11 is the <t>Msp</t> I digest of <t>pBR322.</t> The size of each fragment is noted on the right. The parameters for β 1 negative cells in lane 4: A 1 = 9.80, τ 1 = 13.75, A 2 = 14.17, τ 2 = 64.61, τ DW = 43.82l; and lane 5: A 1 = 12.94, τ 1 = 12.94, A 2 = 5.69, τ 2 = 77.21, τ DW = 29.63. The β 1 positive cell in lane 6: A 1 = 18.05, τ 1 = 15.11, A 2 = 4.15, τ 2 = 74.44, τ DW = 26.20.
    Pbr322 Dna Msp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cells with fast initial decay kinetics express β 1 subunit mRNA A and B , scatter plots of IPSC 90%-width versus peak current amplitude for two RTN cells recorded on the same day. Each point in A and B represents an individual IPSC. A is a recording from a cell dominated with slow and small IPSCs (○) and B , represents a recording dominated by brief and large IPSCs ( ). C , ensemble averaged IPSCs averaged from isolated spontaneous inhibitory events (selected on the basis of having arisen from an event-free baseline and showing complete decay to the same baseline) for the two cells represented in A (black, n = 258) and B (grey, n = 217). Each trace was best fitted to two exponentials (dotted line superimposed on each trace). Note: the cell dominated with fast IPSCs yielded a ensemble averaged IPSC with a larger A 1 value than A 2 whereas the opposite is true for the slower cell. D , ethidium bromide stained 8% polyacrylamide gel showing single-cell RT-PCR analysis from five RTN cells examined in one day of recording, two of the cells are represented in A (lane 3) and B (lane 7). Cells positive for the β 1 subunit cDNA showed a solitary band at 162 bp after two rounds of amplification using nested primers (lanes 6 and 7, see methods). Lanes 1 and 2 are water and media controls that underwent reverse transcription along with individual cells in lanes 3 through 7; lane 8 is a positive control using 40 pg of total RNA; lanes 9 and 10 are water controls used in the master mix for each round of PCR amplification. The molecular weight marker in lane 11 is the Msp I digest of pBR322. The size of each fragment is noted on the right. The parameters for β 1 negative cells in lane 4: A 1 = 9.80, τ 1 = 13.75, A 2 = 14.17, τ 2 = 64.61, τ DW = 43.82l; and lane 5: A 1 = 12.94, τ 1 = 12.94, A 2 = 5.69, τ 2 = 77.21, τ DW = 29.63. The β 1 positive cell in lane 6: A 1 = 18.05, τ 1 = 15.11, A 2 = 4.15, τ 2 = 74.44, τ DW = 26.20.

    Journal: The Journal of Physiology

    Article Title: Fast IPSCs in rat thalamic reticular nucleus require the GABAA receptor ?1 subunit

    doi: 10.1113/jphysiol.2006.106617

    Figure Lengend Snippet: Cells with fast initial decay kinetics express β 1 subunit mRNA A and B , scatter plots of IPSC 90%-width versus peak current amplitude for two RTN cells recorded on the same day. Each point in A and B represents an individual IPSC. A is a recording from a cell dominated with slow and small IPSCs (○) and B , represents a recording dominated by brief and large IPSCs ( ). C , ensemble averaged IPSCs averaged from isolated spontaneous inhibitory events (selected on the basis of having arisen from an event-free baseline and showing complete decay to the same baseline) for the two cells represented in A (black, n = 258) and B (grey, n = 217). Each trace was best fitted to two exponentials (dotted line superimposed on each trace). Note: the cell dominated with fast IPSCs yielded a ensemble averaged IPSC with a larger A 1 value than A 2 whereas the opposite is true for the slower cell. D , ethidium bromide stained 8% polyacrylamide gel showing single-cell RT-PCR analysis from five RTN cells examined in one day of recording, two of the cells are represented in A (lane 3) and B (lane 7). Cells positive for the β 1 subunit cDNA showed a solitary band at 162 bp after two rounds of amplification using nested primers (lanes 6 and 7, see methods). Lanes 1 and 2 are water and media controls that underwent reverse transcription along with individual cells in lanes 3 through 7; lane 8 is a positive control using 40 pg of total RNA; lanes 9 and 10 are water controls used in the master mix for each round of PCR amplification. The molecular weight marker in lane 11 is the Msp I digest of pBR322. The size of each fragment is noted on the right. The parameters for β 1 negative cells in lane 4: A 1 = 9.80, τ 1 = 13.75, A 2 = 14.17, τ 2 = 64.61, τ DW = 43.82l; and lane 5: A 1 = 12.94, τ 1 = 12.94, A 2 = 5.69, τ 2 = 77.21, τ DW = 29.63. The β 1 positive cell in lane 6: A 1 = 18.05, τ 1 = 15.11, A 2 = 4.15, τ 2 = 74.44, τ DW = 26.20.

    Article Snippet: The molecular weight marker was the pBR322 DNA– Msp I digest (New England Biolabs, Ipswich, MA, USA).

    Techniques: Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control, Polymerase Chain Reaction, Molecular Weight, Marker

    Time course of in vitro Pol II transcription through a nucleosome in the absence/presence of human FACT ], with permission). The in vitro Pol II transcription through the 603 nucleosome in the absence or presence of hFACT was conducted at 150 mM KCl for indicated time intervals (0, 5, 10, 15, 20, 25, 30, 60 seconds). After reactions, the pulse-labeled RNA was purified and analyzed by denaturing PAGE. The image was then obtained using phosphoimager and analyzed using OptiQuant software. The position of the nucleosome on the DNA template is indicated. The bands corresponding to nucleosome-specific pausing sites are quantified. The quantified bands are then grouped into 10 clusters of the pausing intermediates (A to J) and the run-off (K). M: pBR322 DNA- Msp I digest.

    Journal: Methods (San Diego, Calif.)

    Article Title: Time-Resolved Analysis of Transcription through Chromatin

    doi: 10.1016/j.ymeth.2019.01.016

    Figure Lengend Snippet: Time course of in vitro Pol II transcription through a nucleosome in the absence/presence of human FACT ], with permission). The in vitro Pol II transcription through the 603 nucleosome in the absence or presence of hFACT was conducted at 150 mM KCl for indicated time intervals (0, 5, 10, 15, 20, 25, 30, 60 seconds). After reactions, the pulse-labeled RNA was purified and analyzed by denaturing PAGE. The image was then obtained using phosphoimager and analyzed using OptiQuant software. The position of the nucleosome on the DNA template is indicated. The bands corresponding to nucleosome-specific pausing sites are quantified. The quantified bands are then grouped into 10 clusters of the pausing intermediates (A to J) and the run-off (K). M: pBR322 DNA- Msp I digest.

    Article Snippet: Salmon sperm dsDNA (Sigma). pBR322 DNA- MspI Digest (New England Biolabs).

    Techniques: In Vitro, Labeling, Purification, Polyacrylamide Gel Electrophoresis, Software

    Competition between H-NS and SlyA binding upstream of the fimB promoter. A , binding of H-NS to the fim03 fragment labeled at the fim03f end. Decreasing concentrations of H-NS were mixed with the fim03 DNA with and without SlyA in the “HNS” buffer. Lane 1 , no proteins; lanes 2 and 7 , 400 n m H-NS; lanes 3 and 8 , 200 n m H-NS; lanes 4 and 9 , 100 n m H-NS; lanes 5 and 10 , 50 n m H-NS; lanes 6 and 12 , 25 n m H-NS; lanes 7–12 also contained 2 μ m SlyA. B , H-NS binding with and without SlyA in the “SlyA” buffer. Lane 1 , no proteins; lane 2 , 50 n m SlyA; lane 3 , 1 μ m SlyA; lane 4 , 50 n m H-NS; lane 5 , 100 n m H-NS; lane 6 , 1 μ m SlyA, and 50 n m H-NS; lane 7, 1 μ m SlyA and 100 n m H-NS. Proteins were incubated for 15 min at 25 °C before treatment with DNase I. Products were analyzed on 6% denaturing polyacrylamide gels. Regions protected by H-NS and SlyA are indicated. The arrows indicate hypersensitive DNaseI cleavages on the DNA in the presence of H-NS, which are observed in the “SlyA” buffer ( B ) but not in the “H-NS” buffer (H-NS). The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Journal: The Journal of Biological Chemistry

    Article Title: SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12 *

    doi: 10.1074/jbc.M111.266619

    Figure Lengend Snippet: Competition between H-NS and SlyA binding upstream of the fimB promoter. A , binding of H-NS to the fim03 fragment labeled at the fim03f end. Decreasing concentrations of H-NS were mixed with the fim03 DNA with and without SlyA in the “HNS” buffer. Lane 1 , no proteins; lanes 2 and 7 , 400 n m H-NS; lanes 3 and 8 , 200 n m H-NS; lanes 4 and 9 , 100 n m H-NS; lanes 5 and 10 , 50 n m H-NS; lanes 6 and 12 , 25 n m H-NS; lanes 7–12 also contained 2 μ m SlyA. B , H-NS binding with and without SlyA in the “SlyA” buffer. Lane 1 , no proteins; lane 2 , 50 n m SlyA; lane 3 , 1 μ m SlyA; lane 4 , 50 n m H-NS; lane 5 , 100 n m H-NS; lane 6 , 1 μ m SlyA, and 50 n m H-NS; lane 7, 1 μ m SlyA and 100 n m H-NS. Proteins were incubated for 15 min at 25 °C before treatment with DNase I. Products were analyzed on 6% denaturing polyacrylamide gels. Regions protected by H-NS and SlyA are indicated. The arrows indicate hypersensitive DNaseI cleavages on the DNA in the presence of H-NS, which are observed in the “SlyA” buffer ( B ) but not in the “H-NS” buffer (H-NS). The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Article Snippet: The size marker is pBR322 DNA digested with MspI (New England Biolabs) and labeled with [γ-32 P]ATP and polynucleotide kinase.

    Techniques: Binding Assay, Labeling, Incubation, Marker

    The interaction of SlyA with the fimB promoter region in vitro . A , effect of 0, 15, 30, 60, and 120 n m SlyA dimer on the electophoretic mobility of DNA amplicons fim03 and pBS (each 11 n m ). Amplicon fim03 is 282 bp of the region upstream of fimB and includes O SA1 and O SA2 ( Fig. 1 ). Amplicon pBS is a negative control as previously described ( 27 ). The SlyA-DNA complex is indicated with an arrow . Samples were separated on a 5% polyacrylamide gel. Electrophoresis was carried out at 160 V for 35 min as described under “Experimental Procedures.” B , DNase I footprinting. The fim03 fragment, labeled at the fim03r end, was mixed with decreasing concentrations of SlyA for 15 min at 25 °C before digestion with DNaseI. Lane 1 , no SlyA; lane 2 , 1 μ m SlyA; lane 3 , 500 n m ; lane 4 , 250 n m ; lane 5 , 125 n m ; lane 6 , 62.5 n m ; lane 7 , 31 n m ; lane 8 , 15.6 n m . The products were analyzed on a 6% denaturing polyacrylamide gel. Regions protected by SlyA are indicated. The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Journal: The Journal of Biological Chemistry

    Article Title: SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12 *

    doi: 10.1074/jbc.M111.266619

    Figure Lengend Snippet: The interaction of SlyA with the fimB promoter region in vitro . A , effect of 0, 15, 30, 60, and 120 n m SlyA dimer on the electophoretic mobility of DNA amplicons fim03 and pBS (each 11 n m ). Amplicon fim03 is 282 bp of the region upstream of fimB and includes O SA1 and O SA2 ( Fig. 1 ). Amplicon pBS is a negative control as previously described ( 27 ). The SlyA-DNA complex is indicated with an arrow . Samples were separated on a 5% polyacrylamide gel. Electrophoresis was carried out at 160 V for 35 min as described under “Experimental Procedures.” B , DNase I footprinting. The fim03 fragment, labeled at the fim03r end, was mixed with decreasing concentrations of SlyA for 15 min at 25 °C before digestion with DNaseI. Lane 1 , no SlyA; lane 2 , 1 μ m SlyA; lane 3 , 500 n m ; lane 4 , 250 n m ; lane 5 , 125 n m ; lane 6 , 62.5 n m ; lane 7 , 31 n m ; lane 8 , 15.6 n m . The products were analyzed on a 6% denaturing polyacrylamide gel. Regions protected by SlyA are indicated. The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Article Snippet: The size marker is pBR322 DNA digested with MspI (New England Biolabs) and labeled with [γ-32 P]ATP and polynucleotide kinase.

    Techniques: In Vitro, Amplification, Negative Control, Polyacrylamide Gel Electrophoresis, Footprinting, Labeling, Marker

    DNA-protective activity of skyrin (SK) analyzed by DNA-topology assay. Electrophoretic monitoring of changes induced by FeSO 4 × 7H 2 O in the topology of plasmid DNA (pBR322) after skyrin application (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + FeSO 4 × 7H 2 O + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.

    Journal: International Journal of Molecular Sciences

    Article Title: Comparison of Cytotoxic, Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells

    doi: 10.3390/ijms23105339

    Figure Lengend Snippet: DNA-protective activity of skyrin (SK) analyzed by DNA-topology assay. Electrophoretic monitoring of changes induced by FeSO 4 × 7H 2 O in the topology of plasmid DNA (pBR322) after skyrin application (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + FeSO 4 × 7H 2 O + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.

    Article Snippet: The electrophoretic monitoring of topological changes in the plasmid DNA (pBR322, New England BioLabs, Ipswich, MA, USA) induced by FeSO4 × 7H2 O (Lachema, Brno, Czech republic) was used to detect the DNA-damaging and DNA-protective potential of skyrin, as [ , ] described in detail.

    Techniques: Activity Assay, Plasmid Preparation, Negative Control, Positive Control

    Potential DNA-damaging effects of skyrin on plasmid DNA. Electrophoretic monitoring of changes in the topology of plasmid DNA (pBR322) induced by skyrin (SK) using DNA-topology assay (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.

    Journal: International Journal of Molecular Sciences

    Article Title: Comparison of Cytotoxic, Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells

    doi: 10.3390/ijms23105339

    Figure Lengend Snippet: Potential DNA-damaging effects of skyrin on plasmid DNA. Electrophoretic monitoring of changes in the topology of plasmid DNA (pBR322) induced by skyrin (SK) using DNA-topology assay (Form I = relaxed circular DNA; Form II = linear DNA; Form III = supercoiled DNA). Legend: Line 1: pBR322 (negative control-NC); line 2: pBR322 + FeSO 4 × 7H 2 O (positive control-PC); lines 3–12: pBR322 + different concentrations of skyrin. Experiments were performed three times with similar results, and the representative electrophoretic gel is presented. The bar chart shows the percentage of individual topological forms of plasmid DNA in corresponding lines.

    Article Snippet: The electrophoretic monitoring of topological changes in the plasmid DNA (pBR322, New England BioLabs, Ipswich, MA, USA) induced by FeSO4 × 7H2 O (Lachema, Brno, Czech republic) was used to detect the DNA-damaging and DNA-protective potential of skyrin, as [ , ] described in detail.

    Techniques: Plasmid Preparation, Negative Control, Positive Control

    Evaluation of the potential DNA-protective effects of skyrin (SK) on the HepG2 cell line (24 h treatment) at different concentrations using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 1 mM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (24 h treatment), and hydrogen peroxide (1 mM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p

    Journal: International Journal of Molecular Sciences

    Article Title: Comparison of Cytotoxic, Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells

    doi: 10.3390/ijms23105339

    Figure Lengend Snippet: Evaluation of the potential DNA-protective effects of skyrin (SK) on the HepG2 cell line (24 h treatment) at different concentrations using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 1 mM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (24 h treatment), and hydrogen peroxide (1 mM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p

    Article Snippet: The electrophoretic monitoring of topological changes in the plasmid DNA (pBR322, New England BioLabs, Ipswich, MA, USA) induced by FeSO4 × 7H2 O (Lachema, Brno, Czech republic) was used to detect the DNA-damaging and DNA-protective potential of skyrin, as [ , ] described in detail.

    Techniques: Single Cell Gel Electrophoresis, Negative Control, Positive Control, Concentration Assay

    Evaluation of the potential DNA-protective effects of skyrin (SK) on human lymphocytes (1 h treatment) at different concentrations, using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 440 μM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (1 h treatment), and hydrogen peroxide (440 μM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p

    Journal: International Journal of Molecular Sciences

    Article Title: Comparison of Cytotoxic, Genotoxic, and DNA-Protective Effects of Skyrin on Cancerous vs. Non-Cancerous Human Cells

    doi: 10.3390/ijms23105339

    Figure Lengend Snippet: Evaluation of the potential DNA-protective effects of skyrin (SK) on human lymphocytes (1 h treatment) at different concentrations, using the comet assay. Negative control (NC)—untreated cells; positive control for comet assay (PC)—cells treated with 440 μM hydrogen peroxide. Tested samples were treated with a different concentration of skyrin (1 h treatment), and hydrogen peroxide (440 μM) was subsequently added (5 min treatment). Results were analyzed by ANOVA single-step multiple comparisons of means using LSD tests, and comparisons between the mean values were considered significant at p

    Article Snippet: The electrophoretic monitoring of topological changes in the plasmid DNA (pBR322, New England BioLabs, Ipswich, MA, USA) induced by FeSO4 × 7H2 O (Lachema, Brno, Czech republic) was used to detect the DNA-damaging and DNA-protective potential of skyrin, as [ , ] described in detail.

    Techniques: Single Cell Gel Electrophoresis, Negative Control, Positive Control, Concentration Assay