dnase i  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 77
    Name:
    DNase I
    Description:
    Thermo Scientific DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-OH groups.The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions.In the presence of Mg2+, DNase I cleaves each strand of dsDNA independently in a statistically random fashion. In the presence of Mn2+, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with overhang termini of only one or two nucleotides.Highlights• Recombinant enzyme• Purified from non-animal host with a lower level of intrinsic RNasesApplications• Preparation of DNA-free RNA• Removal of template DNA following in vitro transcription• Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR• DNA labeling by nick-translation in conjunction with DNA Polymerase I• Studies of DNA-protein interactions by DNase I, RNase-free footprinting• Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn2+ is usedNoteDNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not vortex.
    Catalog Number:
    EN0521
    Price:
    None
    Applications:
    In Vitro Transcription|One-Step qRT-PCR|PCR & Real-Time PCR|RT-PCR|Real Time PCR (qPCR)|Reverse Transcription|Two-Step RT-PCR|Gene Expression Analysis & Genotyping
    Size:
    1 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher dnase i
    Mast cells release DNA in response to heat-killed but not live  Mycobacterium tuberculosis  (Mtb).  (A)  Representative micrographs of HMC-1 cells or bone marrow-derived mast cells (BMMC) unstimulated (control) or stimulated during 2 h with PMA, live Mtb, or heat-killed Mtb (HK-Mtb) at a MOI of 10. DNA was visualized after staining with SYTOX-Green. Scale bar 20 µm (magnification 400×).  (B)  Released DNA was quantified in unstimulated HMC-1 cells (control) or stimulated at indicated times with PMA, live Mtb, or HK-Mtb at a MOI of 10. Released DNA was partially digested with DNase I and quantified in supernatants with SYTOX Green I in a fluorometer. The graph represents the change in fluorescence ± SD of stimulated cells compared to control. *** p
    Thermo Scientific DNase I, RNase-free is an endonuclease that digests single- and double-stranded DNA. It hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5'-phosphate and 3'-OH groups.The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions.In the presence of Mg2+, DNase I cleaves each strand of dsDNA independently in a statistically random fashion. In the presence of Mn2+, the enzyme cleaves both DNA strands at approximately the same site, producing DNA fragments with blunt-ends or with overhang termini of only one or two nucleotides.Highlights• Recombinant enzyme• Purified from non-animal host with a lower level of intrinsic RNasesApplications• Preparation of DNA-free RNA• Removal of template DNA following in vitro transcription• Preparation of DNA-free RNA prior to RT-PCR and RT-qPCR• DNA labeling by nick-translation in conjunction with DNA Polymerase I• Studies of DNA-protein interactions by DNase I, RNase-free footprinting• Generation of a library of randomly overlapping DNA inserts. Reaction buffer containing Mn2+ is usedNoteDNase I is sensitive to physical denaturation. Mix gently by inverting the tube. Do not vortex.
    https://www.bioz.com/result/dnase i/product/Thermo Fisher
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-01
    77/100 stars

    Images

    1) Product Images from "Mycobacterium tuberculosis Catalase Inhibits the Formation of Mast Cell Extracellular Traps"

    Article Title: Mycobacterium tuberculosis Catalase Inhibits the Formation of Mast Cell Extracellular Traps

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01161

    Mast cells release DNA in response to heat-killed but not live  Mycobacterium tuberculosis  (Mtb).  (A)  Representative micrographs of HMC-1 cells or bone marrow-derived mast cells (BMMC) unstimulated (control) or stimulated during 2 h with PMA, live Mtb, or heat-killed Mtb (HK-Mtb) at a MOI of 10. DNA was visualized after staining with SYTOX-Green. Scale bar 20 µm (magnification 400×).  (B)  Released DNA was quantified in unstimulated HMC-1 cells (control) or stimulated at indicated times with PMA, live Mtb, or HK-Mtb at a MOI of 10. Released DNA was partially digested with DNase I and quantified in supernatants with SYTOX Green I in a fluorometer. The graph represents the change in fluorescence ± SD of stimulated cells compared to control. *** p
    Figure Legend Snippet: Mast cells release DNA in response to heat-killed but not live Mycobacterium tuberculosis (Mtb). (A) Representative micrographs of HMC-1 cells or bone marrow-derived mast cells (BMMC) unstimulated (control) or stimulated during 2 h with PMA, live Mtb, or heat-killed Mtb (HK-Mtb) at a MOI of 10. DNA was visualized after staining with SYTOX-Green. Scale bar 20 µm (magnification 400×). (B) Released DNA was quantified in unstimulated HMC-1 cells (control) or stimulated at indicated times with PMA, live Mtb, or HK-Mtb at a MOI of 10. Released DNA was partially digested with DNase I and quantified in supernatants with SYTOX Green I in a fluorometer. The graph represents the change in fluorescence ± SD of stimulated cells compared to control. *** p

    Techniques Used: Derivative Assay, Staining, Fluorescence

    2) Product Images from "Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors"

    Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02083-1

    DNA-binding proteins and mitochondrial and apicoplast genes in EVs.  a  PCR for Ev-DNA markers, apicoplast ( ssu-api ), mitochondria ( ssud ) and nuclear genes ( msp2 ,  rap14 , and  gap40 ). Plasmid control PuF-1 was added externally to EVs prior to DNase I treatment.  b P. falciparum msp2  gene Ev-FISH images of ring and trophozoite EVs and uRBC vesicles.  c P. falciparum ssu-api  and  ssud  genes Ev-FISH of ring EVs.  d  H3 and H4 protein WB analysis for 2–5 OP gradient fractions (F).  e P. falciparum  H4 protein IFA.  f P .  falciparum  parasites release EVs during the early post-invasion phase. Fluorescence microscopy using DAPI in EVs produced by iRBCs across their life cycle. Images of EVs collected at 12, 24, 36 and 48 h post invasion. Giemsa stains (first column) show the state of the parasites prior to collecting EVs at each time point.  g  SR1 control IFA
    Figure Legend Snippet: DNA-binding proteins and mitochondrial and apicoplast genes in EVs. a PCR for Ev-DNA markers, apicoplast ( ssu-api ), mitochondria ( ssud ) and nuclear genes ( msp2 , rap14 , and gap40 ). Plasmid control PuF-1 was added externally to EVs prior to DNase I treatment. b P. falciparum msp2 gene Ev-FISH images of ring and trophozoite EVs and uRBC vesicles. c P. falciparum ssu-api and ssud genes Ev-FISH of ring EVs. d H3 and H4 protein WB analysis for 2–5 OP gradient fractions (F). e P. falciparum H4 protein IFA. f P . falciparum parasites release EVs during the early post-invasion phase. Fluorescence microscopy using DAPI in EVs produced by iRBCs across their life cycle. Images of EVs collected at 12, 24, 36 and 48 h post invasion. Giemsa stains (first column) show the state of the parasites prior to collecting EVs at each time point. g SR1 control IFA

    Techniques Used: DNA Binding Assay, Polymerase Chain Reaction, Plasmid Preparation, Fluorescence In Situ Hybridization, Western Blot, Immunofluorescence, Fluorescence, Microscopy, Produced

    3) Product Images from "Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum"

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005686

    Histones and DNA contribute to the bactericidal activity of pea root border cells. (A) Histone H4, a component of the root cap secretome, is bactericidal to  R .  solanacearum . Percent live bacteria in the presence of histone H4 was determined by the BacLight LIVE/DEAD staining kit. A standard curve of percentage of live cells relative to SYTO9/PI fluorescence intensity was constructed using known ratios of live/dead bacteria (R 2  = 0.99). The experiment was repeated twice, each with three technical replicates. (B) Bactericidal activity of root border cells on  R .  solanacearum . This effect was blocked by addition of either anti-Histone H4 antibody or DNase I. Asterisks indicate treatment significantly different from the bacteria-only control (one-way ANOVA, **** p
    Figure Legend Snippet: Histones and DNA contribute to the bactericidal activity of pea root border cells. (A) Histone H4, a component of the root cap secretome, is bactericidal to R . solanacearum . Percent live bacteria in the presence of histone H4 was determined by the BacLight LIVE/DEAD staining kit. A standard curve of percentage of live cells relative to SYTO9/PI fluorescence intensity was constructed using known ratios of live/dead bacteria (R 2 = 0.99). The experiment was repeated twice, each with three technical replicates. (B) Bactericidal activity of root border cells on R . solanacearum . This effect was blocked by addition of either anti-Histone H4 antibody or DNase I. Asterisks indicate treatment significantly different from the bacteria-only control (one-way ANOVA, **** p

    Techniques Used: Activity Assay, Staining, Fluorescence, Construct

    R .  solanacearum  genes encode two secreted DNases. (A) DNase activity of cell-free bacterial culture supernatant from  R .  solanacearum  strains grown in minimal medium, measured by DNase Alert assay on a fluorescence plate reader at 37°C over 3 h. Bars represent mean relative fluorescence units normalized to A 600  of overnight culture. (B) Activity of purified nucleases on different DNA substrates. 1 μg of purified NucA or NucB were incubated with 1 μg of each DNA substrate:  R .  solanacearum  genomic DNA, pea DNA, supercoiled plasmid DNA (pUCGM) and salmon sperm DNA) for 30 min at 37°C. Results were analyzed by electrophoresis in a 1% agarose gel. (C) NucA and NucB degrade DNA from root border cell traps. Pea border cells were incubated with  R .  solanacearum  in the presence of NucA, NucB or DNase I as control. Relative DNA amount was measured by SYTOX Green fluorescence after 6 h of incubation. Asterisks indicate differences from the wild-type (one-way ANOVA, * P
    Figure Legend Snippet: R . solanacearum genes encode two secreted DNases. (A) DNase activity of cell-free bacterial culture supernatant from R . solanacearum strains grown in minimal medium, measured by DNase Alert assay on a fluorescence plate reader at 37°C over 3 h. Bars represent mean relative fluorescence units normalized to A 600 of overnight culture. (B) Activity of purified nucleases on different DNA substrates. 1 μg of purified NucA or NucB were incubated with 1 μg of each DNA substrate: R . solanacearum genomic DNA, pea DNA, supercoiled plasmid DNA (pUCGM) and salmon sperm DNA) for 30 min at 37°C. Results were analyzed by electrophoresis in a 1% agarose gel. (C) NucA and NucB degrade DNA from root border cell traps. Pea border cells were incubated with R . solanacearum in the presence of NucA, NucB or DNase I as control. Relative DNA amount was measured by SYTOX Green fluorescence after 6 h of incubation. Asterisks indicate differences from the wild-type (one-way ANOVA, * P

    Techniques Used: Activity Assay, Fluorescence, Purification, Incubation, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis

    4) Product Images from "Reconstitution of 'floral quartets' in vitro involving class B and class E floral homeotic proteins"

    Article Title: Reconstitution of 'floral quartets' in vitro involving class B and class E floral homeotic proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp129

    Formation of SEP3-SEP3/AP3-PI complexes. As in   Figure 2 , bands are marked with numbers (‘0’, ‘2’ and ‘4’) according to the supposed number of proteins bound to the DNA fragment. ‘4#’ denotes bands supposed to contain SEP3 and AP3-PI bound to the same DNA fragment. ‘M’ denotes marker lanes. DNA probes are symbolized with icons as in Figure 1. ( A ) Formation of SEP3 homotetramers. Probes in which the phasing between two  AG -derived CArG boxes varied as noted aboved the gel were co-incubated with 0.4 µl of  in vitro  translated SEP3. A SEP3 dimer bound to probe C is shown for comparison in lane 3. ‘Δ’ denotes a negative control in which the  in vitro  translation assay was programmed only by empty pTNT vector and co-incubated with probe A. ( B ) EMSA experiment in which SEP3 and/or AP3-PI were incubated with probe A. Proteins that were radioactively labelled with  35 S methionine during the  in vitro  translation are marked by asterisks. When at least one of the proteins was labelled, unlabelled DNA fragments were used, otherwise the DNA-fragment was labelled. The binding cocktail contained 1.5 µl of  in vitro  translated protein or, in cases where SEP3 was incubated with AP3-PI, 1.5 µl of each of the two  in vitro  translations. All labelled or unlabelled proteins of the same type were taken from the same  in vitro  translation reaction and thus had the same activity. ‘Δ’ denotes a negative control in which empty pSPUTK vector was applied ( in vitro  translated with  35 S methionine and incubated with unlabelled DNA). ( C ) Phase dependence of the formation of a DNA bound SEP3-SEP3/AP3-PI complex. The binding cocktail contained 0.5 µl of  in vitro  translated protein. SEP3, AP3 and PI were co-translated in a single reaction. Signals resulting from complexes bound to probes B and C are shown in the leftmost lanes. The other probes contained two copies of the  AG -derived CArG box (probe A in lane 3). Phasing between the CArG boxes was varied as noted aboved the gel. On the right of the gel picture, quantitative analysis of homo- and heterotetramer formation is shown. Fractional saturation of the signal intensity caused by the different tetramers is expressed as percentage of the fractional saturation of the homo- or heterotetramers bound to probe A (lane 3). ( D ) DNase I footprint assays. SEP3 or SEP3 +AP3-PI was incubated with probe A. SEP3 and AP3-PI were separately translated and equal amounts were mixed. SEP3 preparations used in the left and right part of the figure were from different  in vitro  translation assays but the same results were obtained with aliquots. ‘0’, ‘4’ and ‘4#’ indicate protection patterns obtained for free DNA (‘0’) and complexes ‘4’ and ‘4#’ extracted from an EMSA gel. As complexes ‘4’ and ‘4#’ migrate closely together, it was not possible to excise them separately. To obtain the ‘free DNA’ pattern, the DNA probe was incubated with an  in vitro  translation reaction that contained empty pTNT vector. An A+G sequencing reaction of the DNA probe is shown for comparison. Sequence of the DNA is depicted on the right (blue = cytosine, red = thymine, green = adenine, black = guanine); the position of the CArG boxes is indicated. Open and filled arrowheads point towards sites of diminished and enhanced DNase I sensitivity, respectively, after binding of protein. Beneath the gel picture, quantitative analysis of the DNAse I footprint assays shows the change of sensitivity to DNase I digestion after protein binding in single base pair-steps, using free DNA as a reference. Values were corrected for differences in DNA loading by using invariable internal bands as a reference and represent the mean of two experiments.
    Figure Legend Snippet: Formation of SEP3-SEP3/AP3-PI complexes. As in Figure 2 , bands are marked with numbers (‘0’, ‘2’ and ‘4’) according to the supposed number of proteins bound to the DNA fragment. ‘4#’ denotes bands supposed to contain SEP3 and AP3-PI bound to the same DNA fragment. ‘M’ denotes marker lanes. DNA probes are symbolized with icons as in Figure 1. ( A ) Formation of SEP3 homotetramers. Probes in which the phasing between two AG -derived CArG boxes varied as noted aboved the gel were co-incubated with 0.4 µl of in vitro translated SEP3. A SEP3 dimer bound to probe C is shown for comparison in lane 3. ‘Δ’ denotes a negative control in which the in vitro translation assay was programmed only by empty pTNT vector and co-incubated with probe A. ( B ) EMSA experiment in which SEP3 and/or AP3-PI were incubated with probe A. Proteins that were radioactively labelled with 35 S methionine during the in vitro translation are marked by asterisks. When at least one of the proteins was labelled, unlabelled DNA fragments were used, otherwise the DNA-fragment was labelled. The binding cocktail contained 1.5 µl of in vitro translated protein or, in cases where SEP3 was incubated with AP3-PI, 1.5 µl of each of the two in vitro translations. All labelled or unlabelled proteins of the same type were taken from the same in vitro translation reaction and thus had the same activity. ‘Δ’ denotes a negative control in which empty pSPUTK vector was applied ( in vitro translated with 35 S methionine and incubated with unlabelled DNA). ( C ) Phase dependence of the formation of a DNA bound SEP3-SEP3/AP3-PI complex. The binding cocktail contained 0.5 µl of in vitro translated protein. SEP3, AP3 and PI were co-translated in a single reaction. Signals resulting from complexes bound to probes B and C are shown in the leftmost lanes. The other probes contained two copies of the AG -derived CArG box (probe A in lane 3). Phasing between the CArG boxes was varied as noted aboved the gel. On the right of the gel picture, quantitative analysis of homo- and heterotetramer formation is shown. Fractional saturation of the signal intensity caused by the different tetramers is expressed as percentage of the fractional saturation of the homo- or heterotetramers bound to probe A (lane 3). ( D ) DNase I footprint assays. SEP3 or SEP3 +AP3-PI was incubated with probe A. SEP3 and AP3-PI were separately translated and equal amounts were mixed. SEP3 preparations used in the left and right part of the figure were from different in vitro translation assays but the same results were obtained with aliquots. ‘0’, ‘4’ and ‘4#’ indicate protection patterns obtained for free DNA (‘0’) and complexes ‘4’ and ‘4#’ extracted from an EMSA gel. As complexes ‘4’ and ‘4#’ migrate closely together, it was not possible to excise them separately. To obtain the ‘free DNA’ pattern, the DNA probe was incubated with an in vitro translation reaction that contained empty pTNT vector. An A+G sequencing reaction of the DNA probe is shown for comparison. Sequence of the DNA is depicted on the right (blue = cytosine, red = thymine, green = adenine, black = guanine); the position of the CArG boxes is indicated. Open and filled arrowheads point towards sites of diminished and enhanced DNase I sensitivity, respectively, after binding of protein. Beneath the gel picture, quantitative analysis of the DNAse I footprint assays shows the change of sensitivity to DNase I digestion after protein binding in single base pair-steps, using free DNA as a reference. Values were corrected for differences in DNA loading by using invariable internal bands as a reference and represent the mean of two experiments.

    Techniques Used: Marker, Derivative Assay, Incubation, In Vitro, Negative Control, Plasmid Preparation, Binding Assay, Activity Assay, Sequencing, Protein Binding

    Model of a floral quartet. ( A ) B DNA of which roll angles have been changed according to the differences of sensitivity to DNase I digestion of SEP3-SEP3/AP3-PI bound DNA versus free DNA (see ‘Materials and Methods’ section). The CArG boxes on one strand are shown in orange. ( B ) The DNA shown in (A) is modified so that the MADS domain structure of SRF can be plotted on it to infer the relative orientation of the proteins. The original CArG boxes have been replaced by the bent CArG boxes from the SRF–CArG complex (see ‘Materials and Methods’ section). Possible positions of the I and K domains are shown schematically. The C terminus is omitted for simplicity.
    Figure Legend Snippet: Model of a floral quartet. ( A ) B DNA of which roll angles have been changed according to the differences of sensitivity to DNase I digestion of SEP3-SEP3/AP3-PI bound DNA versus free DNA (see ‘Materials and Methods’ section). The CArG boxes on one strand are shown in orange. ( B ) The DNA shown in (A) is modified so that the MADS domain structure of SRF can be plotted on it to infer the relative orientation of the proteins. The original CArG boxes have been replaced by the bent CArG boxes from the SRF–CArG complex (see ‘Materials and Methods’ section). Possible positions of the I and K domains are shown schematically. The C terminus is omitted for simplicity.

    Techniques Used: Modification

    5) Product Images from "Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471"

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076068

    Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of  mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    Figure Legend Snippet: Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Techniques Used: Binding Assay, Footprinting, Labeling, Sequencing, Purification

    Gene expression analysis of the  mur  genes. (A) Transcription analysis of intergenic region of the selected  mur  genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions.  mur10 ← mur11  means that the detected region between  mur10  and  mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of  mur11  and  mur27  for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.
    Figure Legend Snippet: Gene expression analysis of the mur genes. (A) Transcription analysis of intergenic region of the selected mur genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. mur10 ← mur11 means that the detected region between mur10 and mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of mur11 and mur27 for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Techniques Used: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control, Incubation

    6) Product Images from "Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici"

    Article Title: Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici

    Journal: Fungal Genetics and Biology

    doi: 10.1016/j.fgb.2015.03.023

    Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see  Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene  mcs1  (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases  rab7  (815 bp) and  rab11  (807 bp) (see  Table 1 ,  rab7 : primers SK-Sep-63 and SK-Sep-64;  rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp;  rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.
    Figure Legend Snippet: Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene mcs1 (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases rab7 (815 bp) and rab11 (807 bp) (see Table 1 , rab7 : primers SK-Sep-63 and SK-Sep-64; rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp; rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.

    Techniques Used: Polymerase Chain Reaction, Amplification, Purification, Sequencing, Generated, Produced, DNA Sequencing

    7) Product Images from "Evolutionary expansion of a regulatory network by counter-silencing"

    Article Title: Evolutionary expansion of a regulatory network by counter-silencing

    Journal: Nature communications

    doi: 10.1038/ncomms6270

    DNase I DDFA of the  pagC  promoter region In vitro  DNase I footprinting studies were performed on the  pagC  promoter region with H-NS, SlyA, and PhoP-P, at concentrations of 600 nM, 100 nM, and 500 nM respectively, as indicated. DNA-protein complexes were incubated at room temperature before digestion with DNase I. Results are presented as DDFA plots, representing the difference in fluorescent peak height (RFU) between the protein-free control and the experimental sample ( a ). DDFA plots are also shown for the H-NS + SlyA, H NS + PhoP-P, and the H-NS + SlyA + PhoP-P reactions, representing the difference between the H-NS control and the experimental sample ( b ). The relative distance in base pairs to the TSS is indicated on the horizontal axis. Peaks indicate regions of hypersensitivity, typical of bent or distorted DNA, whereas valleys indicate protected regions, typical of protein binding sites. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See  Supplementary Fig. 5  for representative raw chromatograms.
    Figure Legend Snippet: DNase I DDFA of the pagC promoter region In vitro DNase I footprinting studies were performed on the pagC promoter region with H-NS, SlyA, and PhoP-P, at concentrations of 600 nM, 100 nM, and 500 nM respectively, as indicated. DNA-protein complexes were incubated at room temperature before digestion with DNase I. Results are presented as DDFA plots, representing the difference in fluorescent peak height (RFU) between the protein-free control and the experimental sample ( a ). DDFA plots are also shown for the H-NS + SlyA, H NS + PhoP-P, and the H-NS + SlyA + PhoP-P reactions, representing the difference between the H-NS control and the experimental sample ( b ). The relative distance in base pairs to the TSS is indicated on the horizontal axis. Peaks indicate regions of hypersensitivity, typical of bent or distorted DNA, whereas valleys indicate protected regions, typical of protein binding sites. Approximate sizes of peaks of note are indicated. Data represent the mean ± SD; n = 3. See Supplementary Fig. 5 for representative raw chromatograms.

    Techniques Used: In Vitro, Footprinting, Incubation, Protein Binding

    8) Product Images from "Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability"

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02429

    Effect of GSH, DNase I, ciprofloxacin and combination therapy (CT) on  P. aeruginosa  biofilm.  (A)  The resazurin bacterial viability assay showed effect of ciprofloxacin, GSH, DNase I and combination treatment on 48 h old established biofilms of different  P. aeruginosa  CF isolates grown in LB media. All isolates showed significant decrease in bacterial viability when treated with ciprofloxacin (Cip) at the highest concentrations (10 and 15 μg/mL). Conversely, DNase I or GSH-alone treatment and in combination with low Cip concentration (5 μg/mL), resulted in a significant decrease in  P. aeruginosa  viability. The most significant decrease in bacterial viability was observed with biofilms subjected to all three components of the CT GSH + DNase + Cip 5 μg/ml.  (B–I)  Confocal microscopy imaging showing the effect of ciprofloxacin, GSH, DNase I and GSH-combination treatment, on 48 h old established biofilms of AES-1R grown in ASMDM. With untreated AES-1R biofilm  (B) , ciprofloxacin alone did not disrupt the biofilm but showed an increase in red-staining (dead) cells  (C) . Disruption of the biofilm architecture of AES-1R was observed when it was treated with DNase I  (D)  or DNase I combined with ciprofloxacin  (E) . Similarly, GSH alone  (F)  and in combination with either DNase I  (G) , or ciprofloxacin  (H) , increased biofilm disruption and dead bacterial cells. Combination therapy (GSH + DNase I + Cip 5 μg/mL) resulted in complete disruption of the biofilm (I). Scale bar = 50 μm.  (A) ∗ P
    Figure Legend Snippet: Effect of GSH, DNase I, ciprofloxacin and combination therapy (CT) on P. aeruginosa biofilm. (A) The resazurin bacterial viability assay showed effect of ciprofloxacin, GSH, DNase I and combination treatment on 48 h old established biofilms of different P. aeruginosa CF isolates grown in LB media. All isolates showed significant decrease in bacterial viability when treated with ciprofloxacin (Cip) at the highest concentrations (10 and 15 μg/mL). Conversely, DNase I or GSH-alone treatment and in combination with low Cip concentration (5 μg/mL), resulted in a significant decrease in P. aeruginosa viability. The most significant decrease in bacterial viability was observed with biofilms subjected to all three components of the CT GSH + DNase + Cip 5 μg/ml. (B–I) Confocal microscopy imaging showing the effect of ciprofloxacin, GSH, DNase I and GSH-combination treatment, on 48 h old established biofilms of AES-1R grown in ASMDM. With untreated AES-1R biofilm (B) , ciprofloxacin alone did not disrupt the biofilm but showed an increase in red-staining (dead) cells (C) . Disruption of the biofilm architecture of AES-1R was observed when it was treated with DNase I (D) or DNase I combined with ciprofloxacin (E) . Similarly, GSH alone (F) and in combination with either DNase I (G) , or ciprofloxacin (H) , increased biofilm disruption and dead bacterial cells. Combination therapy (GSH + DNase I + Cip 5 μg/mL) resulted in complete disruption of the biofilm (I). Scale bar = 50 μm. (A) ∗ P

    Techniques Used: Viability Assay, Concentration Assay, Confocal Microscopy, Imaging, Staining

    Pyocyanin and eDNA production and biofilm formation by clinical and laboratory strains of  Pseudomonas aeruginosa .  (A)  Pyocyanin production by  P. aeruginosa  cystic fibrosis isolates: the isogens AES-1R and AES-1M (acute and chronic, respectively); AES-2, LESB58 and LES431 and laboratory strains (PA14 WT, PA14Δ phzA-G , DKN370, PAO1WT). Among clinical isolates AES-1M expressed the least pyocyanin (ca. 5 μM), whereas the AES-2 isolate expressed the most (ca. 130 μM). Amongst laboratory strains, the phenazine deficient mutant (PA14Δ phzA-G ) did not express pyocyanin while the pyocyanin overproducing strain (DKN 370) recorded ∼155 μM pyocyanin. Pyocyanin concentration was measured using standard pyocyanin concentration (μM) vs. 691 nm (OD 691 nm ).  (B)  eDNA production, quantified  P. aeruginosa  supernatants from cystic fibrosis (CF) isolates and pyocyanin producing strains showed a significant increase in eDNA production (17–19 μg/mL) in comparison to AES-1M (6 μg/mL).  (C)  Crystal violet assay showing biofilm biomass of  P. aeruginosa  laboratory and clinical strains grown in presence or absence of glutathione (GSH), DNase I, exogenous pyocyanin and exogenous DNA to complement DNase I activity. Strains deficient of pyocyanin production formed significantly smaller biofilm biomasses, whereas addition of pyocyanin (200 μM) and DNA (1 ng/μL) facilitated a significant increase in biofilm biomass. DNase I or GSH-treatment of PA14, pyocyanin over-producing strain DKN 370 and the clinical isolates AES-1R and AES-2 resulted in biofilms similar in size to those of PA14Δ phzA-G  and AES-1M.  (A) ∗ P
    Figure Legend Snippet: Pyocyanin and eDNA production and biofilm formation by clinical and laboratory strains of Pseudomonas aeruginosa . (A) Pyocyanin production by P. aeruginosa cystic fibrosis isolates: the isogens AES-1R and AES-1M (acute and chronic, respectively); AES-2, LESB58 and LES431 and laboratory strains (PA14 WT, PA14Δ phzA-G , DKN370, PAO1WT). Among clinical isolates AES-1M expressed the least pyocyanin (ca. 5 μM), whereas the AES-2 isolate expressed the most (ca. 130 μM). Amongst laboratory strains, the phenazine deficient mutant (PA14Δ phzA-G ) did not express pyocyanin while the pyocyanin overproducing strain (DKN 370) recorded ∼155 μM pyocyanin. Pyocyanin concentration was measured using standard pyocyanin concentration (μM) vs. 691 nm (OD 691 nm ). (B) eDNA production, quantified P. aeruginosa supernatants from cystic fibrosis (CF) isolates and pyocyanin producing strains showed a significant increase in eDNA production (17–19 μg/mL) in comparison to AES-1M (6 μg/mL). (C) Crystal violet assay showing biofilm biomass of P. aeruginosa laboratory and clinical strains grown in presence or absence of glutathione (GSH), DNase I, exogenous pyocyanin and exogenous DNA to complement DNase I activity. Strains deficient of pyocyanin production formed significantly smaller biofilm biomasses, whereas addition of pyocyanin (200 μM) and DNA (1 ng/μL) facilitated a significant increase in biofilm biomass. DNase I or GSH-treatment of PA14, pyocyanin over-producing strain DKN 370 and the clinical isolates AES-1R and AES-2 resulted in biofilms similar in size to those of PA14Δ phzA-G and AES-1M. (A) ∗ P

    Techniques Used: Mutagenesis, Concentration Assay, Crystal Violet Assay, Activity Assay

    9) Product Images from "Inhibition of Heat Shock Transcription Factor Binding by a Linear Polyamide Binding in an Unusual 1:1 Mode"

    Article Title: Inhibition of Heat Shock Transcription Factor Binding by a Linear Polyamide Binding in an Unusual 1:1 Mode

    Journal:

    doi: 10.1002/cbic.201100524

    DNase I footprinting of the N→C linear polyamide library P1–P8. Polyamide concentration increases from left to right at 0, 0.1, 1, 10, 50, 100, 200, 300, 400 and 500 nM. Lane C is a control lane containing only DNA and 500 nM polyamide
    Figure Legend Snippet: DNase I footprinting of the N→C linear polyamide library P1–P8. Polyamide concentration increases from left to right at 0, 0.1, 1, 10, 50, 100, 200, 300, 400 and 500 nM. Lane C is a control lane containing only DNA and 500 nM polyamide

    Techniques Used: Footprinting, Concentration Assay

    DNase I footprinting of the hairpin polyamide library. A) Denaturing polyacrylamide gel for H0 that is representative of H2 and H3. The concentration of polyamide increased from left to right: 0, 10, 50, 100, 500 pM, 1, 5, 10, 50, 100, 500 nM, 1µM.
    Figure Legend Snippet: DNase I footprinting of the hairpin polyamide library. A) Denaturing polyacrylamide gel for H0 that is representative of H2 and H3. The concentration of polyamide increased from left to right: 0, 10, 50, 100, 500 pM, 1, 5, 10, 50, 100, 500 nM, 1µM.

    Techniques Used: Footprinting, Concentration Assay

    Analysis of the DNase I footprints of P9–P11 at HSE3 and HSE4. Because the HSE3/4 site is pallindromic, the linear polyamides are shown aligned in C→N orientations with respect to the GG-containing strand of the target sequence. The mismatch
    Figure Legend Snippet: Analysis of the DNase I footprints of P9–P11 at HSE3 and HSE4. Because the HSE3/4 site is pallindromic, the linear polyamides are shown aligned in C→N orientations with respect to the GG-containing strand of the target sequence. The mismatch

    Techniques Used: Sequencing

    DNase I footprinting of the C→N linear polyamide library. Polyamide concentration increases from left to right at 0, 0.1, 1, 10, 50, 100, 200, 300, 400 and 500 nM. Lane C is a control lane with DNA and 500 nM polyamide and no DNase I. Lane A is
    Figure Legend Snippet: DNase I footprinting of the C→N linear polyamide library. Polyamide concentration increases from left to right at 0, 0.1, 1, 10, 50, 100, 200, 300, 400 and 500 nM. Lane C is a control lane with DNA and 500 nM polyamide and no DNase I. Lane A is

    Techniques Used: Footprinting, Concentration Assay

    10) Product Images from "Concurrent Expression of Latent and a Limited Number of Lytic Genes with Immune Modulation and Antiapoptotic Function by Kaposi's Sarcoma-Associated Herpesvirus Early during Infection of Primary Endothelial and Fibroblast Cells and Subsequent Decline of Lytic Gene Expression"

    Article Title: Concurrent Expression of Latent and a Limited Number of Lytic Genes with Immune Modulation and Antiapoptotic Function by Kaposi's Sarcoma-Associated Herpesvirus Early during Infection of Primary Endothelial and Fibroblast Cells and Subsequent Decline of Lytic Gene Expression

    Journal:

    doi: 10.1128/JVI.78.7.3601-3620.2004

    (A) RT-PCR confirmation of KSHV transcription in HMVEC-d and HFF cells. Total RNA prepared from the infected or uninfected cells was treated with DNase I and amplified by RT-PCR. The products were separated by gel electrophoresis, visualized, and quantified.
    Figure Legend Snippet: (A) RT-PCR confirmation of KSHV transcription in HMVEC-d and HFF cells. Total RNA prepared from the infected or uninfected cells was treated with DNase I and amplified by RT-PCR. The products were separated by gel electrophoresis, visualized, and quantified.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Amplification, Nucleic Acid Electrophoresis

    Kinetics of KSHV ORF 73 and 50 gene expression during primary infection of HMVEC-d (A) and HFF (B) cells. Cells were infected at an MOI of 100 KSHV DNA copies per cell. At different time points p.i. RNA was isolated and treated with DNase I, and 250 ng
    Figure Legend Snippet: Kinetics of KSHV ORF 73 and 50 gene expression during primary infection of HMVEC-d (A) and HFF (B) cells. Cells were infected at an MOI of 100 KSHV DNA copies per cell. At different time points p.i. RNA was isolated and treated with DNase I, and 250 ng

    Techniques Used: Expressing, Infection, Isolation

    11) Product Images from "Single-Stranded DNA Uptake during Gonococcal Transformation"

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation

    Journal:

    doi: 10.1128/JB.00464-16

    Carrying capacities of various DNA fragments in a  nuc  deletion strain. Gonococci were incubated for 1 h with DNA fragments containing a single dye molecule at the center (top, stars). Subsequently, they were treated with DNase I. (a) Probability distribution
    Figure Legend Snippet: Carrying capacities of various DNA fragments in a nuc deletion strain. Gonococci were incubated for 1 h with DNA fragments containing a single dye molecule at the center (top, stars). Subsequently, they were treated with DNase I. (a) Probability distribution

    Techniques Used: Incubation

    dsDUS supports the import of ssDNA. Gonococci (Ng005 Δ pilV ) were incubated for 1 h with DNA fragments containing a single dye molecule at the 5′ end (top, stars). Subsequently, they were treated with DNase I. The fragments consisted of
    Figure Legend Snippet: dsDUS supports the import of ssDNA. Gonococci (Ng005 Δ pilV ) were incubated for 1 h with DNA fragments containing a single dye molecule at the 5′ end (top, stars). Subsequently, they were treated with DNase I. The fragments consisted of

    Techniques Used: Incubation

    12) Product Images from "Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum"

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005686

    Histones and DNA contribute to the bactericidal activity of pea root border cells. (A) Histone H4, a component of the root cap secretome, is bactericidal to  R .  solanacearum . Percent live bacteria in the presence of histone H4 was determined by the BacLight LIVE/DEAD staining kit. A standard curve of percentage of live cells relative to SYTO9/PI fluorescence intensity was constructed using known ratios of live/dead bacteria (R 2  = 0.99). The experiment was repeated twice, each with three technical replicates. (B) Bactericidal activity of root border cells on  R .  solanacearum . This effect was blocked by addition of either anti-Histone H4 antibody or DNase I. Asterisks indicate treatment significantly different from the bacteria-only control (one-way ANOVA, **** p
    Figure Legend Snippet: Histones and DNA contribute to the bactericidal activity of pea root border cells. (A) Histone H4, a component of the root cap secretome, is bactericidal to R . solanacearum . Percent live bacteria in the presence of histone H4 was determined by the BacLight LIVE/DEAD staining kit. A standard curve of percentage of live cells relative to SYTO9/PI fluorescence intensity was constructed using known ratios of live/dead bacteria (R 2 = 0.99). The experiment was repeated twice, each with three technical replicates. (B) Bactericidal activity of root border cells on R . solanacearum . This effect was blocked by addition of either anti-Histone H4 antibody or DNase I. Asterisks indicate treatment significantly different from the bacteria-only control (one-way ANOVA, **** p

    Techniques Used: Activity Assay, Staining, Fluorescence, Construct

    R .  solanacearum  genes encode two secreted DNases. (A) DNase activity of cell-free bacterial culture supernatant from  R .  solanacearum  strains grown in minimal medium, measured by DNase Alert assay on a fluorescence plate reader at 37°C over 3 h. Bars represent mean relative fluorescence units normalized to A 600  of overnight culture. (B) Activity of purified nucleases on different DNA substrates. 1 μg of purified NucA or NucB were incubated with 1 μg of each DNA substrate:  R .  solanacearum  genomic DNA, pea DNA, supercoiled plasmid DNA (pUCGM) and salmon sperm DNA) for 30 min at 37°C. Results were analyzed by electrophoresis in a 1% agarose gel. (C) NucA and NucB degrade DNA from root border cell traps. Pea border cells were incubated with  R .  solanacearum  in the presence of NucA, NucB or DNase I as control. Relative DNA amount was measured by SYTOX Green fluorescence after 6 h of incubation. Asterisks indicate differences from the wild-type (one-way ANOVA, * P
    Figure Legend Snippet: R . solanacearum genes encode two secreted DNases. (A) DNase activity of cell-free bacterial culture supernatant from R . solanacearum strains grown in minimal medium, measured by DNase Alert assay on a fluorescence plate reader at 37°C over 3 h. Bars represent mean relative fluorescence units normalized to A 600 of overnight culture. (B) Activity of purified nucleases on different DNA substrates. 1 μg of purified NucA or NucB were incubated with 1 μg of each DNA substrate: R . solanacearum genomic DNA, pea DNA, supercoiled plasmid DNA (pUCGM) and salmon sperm DNA) for 30 min at 37°C. Results were analyzed by electrophoresis in a 1% agarose gel. (C) NucA and NucB degrade DNA from root border cell traps. Pea border cells were incubated with R . solanacearum in the presence of NucA, NucB or DNase I as control. Relative DNA amount was measured by SYTOX Green fluorescence after 6 h of incubation. Asterisks indicate differences from the wild-type (one-way ANOVA, * P

    Techniques Used: Activity Assay, Fluorescence, Purification, Incubation, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis

    13) Product Images from "A non-canonical multisubunit RNA polymerase encoded by the AR9 phage recognizes the template strand of its uracil-containing promoters"

    Article Title: A non-canonical multisubunit RNA polymerase encoded by the AR9 phage recognizes the template strand of its uracil-containing promoters

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx264

    Promoter binding and promoter opening by AR9 nvRNAP. DNase I footprinting and KMnO 4  probing of nvRNAP complexes with the P077 promoter DNA was performed with DNA templates containing uracil (U) or thymine (T). Positions relative to the TSS (+1) are indicated. Lanes indicated as ‘AG’ show markers. Areas protected from DNase I attack are indicated in blue. A fragment of the P077 promoter sequence is shown below, with uracils that undergo oxidation by KMnO 4  in the presence of AR9 nvRNAP indicated by blue triangles.
    Figure Legend Snippet: Promoter binding and promoter opening by AR9 nvRNAP. DNase I footprinting and KMnO 4 probing of nvRNAP complexes with the P077 promoter DNA was performed with DNA templates containing uracil (U) or thymine (T). Positions relative to the TSS (+1) are indicated. Lanes indicated as ‘AG’ show markers. Areas protected from DNase I attack are indicated in blue. A fragment of the P077 promoter sequence is shown below, with uracils that undergo oxidation by KMnO 4 in the presence of AR9 nvRNAP indicated by blue triangles.

    Techniques Used: Binding Assay, Footprinting, Sequencing

    Functional analysis of the two forms of AR9 nvRNAP. ( A ) Atop: chromatographic profile of AR9 nvRNAP eluted from a MonoQ column with a NaCl concentration gradient. Below: a Coomassie-stained SDS gel of the MonoQ fractions containing five-subunit (5-sub) and four-subunit (4-sub) forms of AR9 nvRNAP. ( B ) Comparison of transcriptional activities of the 5s and 4s nvRNAP. Top panel: RNA extension assay using the RNA-DNA scaffold schematically shown on the left. Below:  in vitro  run-off transcription from the uracil-containing promoter P007 in double- and single-stranded DNA (dsDNA and ssDNA). ‘RO’—a run-off transcript (62 nt for dsDNA and 18 nt for ssDNA). ( C ) DNase I footprinting and KMnO 4  probing of nvRNAP–promoter P077 complexes formed by 5s and 4s nvRNAP. The experiment was performed using the uracil-containing template (with template strand radiolabeled). See Figure   3  legend for details.
    Figure Legend Snippet: Functional analysis of the two forms of AR9 nvRNAP. ( A ) Atop: chromatographic profile of AR9 nvRNAP eluted from a MonoQ column with a NaCl concentration gradient. Below: a Coomassie-stained SDS gel of the MonoQ fractions containing five-subunit (5-sub) and four-subunit (4-sub) forms of AR9 nvRNAP. ( B ) Comparison of transcriptional activities of the 5s and 4s nvRNAP. Top panel: RNA extension assay using the RNA-DNA scaffold schematically shown on the left. Below: in vitro run-off transcription from the uracil-containing promoter P007 in double- and single-stranded DNA (dsDNA and ssDNA). ‘RO’—a run-off transcript (62 nt for dsDNA and 18 nt for ssDNA). ( C ) DNase I footprinting and KMnO 4 probing of nvRNAP–promoter P077 complexes formed by 5s and 4s nvRNAP. The experiment was performed using the uracil-containing template (with template strand radiolabeled). See Figure 3 legend for details.

    Techniques Used: Functional Assay, Concentration Assay, Staining, SDS-Gel, In Vitro, Footprinting

    14) Product Images from "In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease"

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease

    Journal:

    doi:

    Characterization of Dig-labeled riboprobes specific for HCV sense and antisense RNAs. (A) Map location of riboprobes within the HCV genome (see Table    for nucleotide positions). Abbreviations: 1a, HCV genotype 1a; 1b, HCV genotype 1b; nt, nucleotide. (B) Recombinant plasmids containing subgenomic fragments of HCV genotype 1a or 1b were constructed for generation of riboprobes by in vitro transcription from bacteriophage T7 or Sp6 promoters. (C) Dot blot assay for assessing the strand specificity of HCV riboprobes. Unlabeled HCV genomic (sense [S]) and replicative-intermediate (antisense [AS]) target RNAs were synthesized by in vitro transcription from recombinant plasmid pE1-1a, pCORE, or pCU (B), treated with DNase I, and blotted onto nylon membranes. Unlabeled target RNAs were hybridized with Dig-labeled riboprobes synthesized from the genomic (S) or antigenomic (AS) strands of the corresponding recombinant DNA templates. For example, rE1-1a (AS), indicates the antigenomic riboprobe corresponding to the HCV genotype 1a E1 gene. (D) Specificity of positive control riboprobes specific for human beta-actin and HPRT sense RNAs. (E) Specificity of negative control riboprobe for Neo. Unlabeled sense (S) RNA probes and Dig-labeled antisense (AS) RNA probes were generated from recombinant DNA templates containing human and bacterial genes and were hybridized in the dot blot assay.
    Figure Legend Snippet: Characterization of Dig-labeled riboprobes specific for HCV sense and antisense RNAs. (A) Map location of riboprobes within the HCV genome (see Table for nucleotide positions). Abbreviations: 1a, HCV genotype 1a; 1b, HCV genotype 1b; nt, nucleotide. (B) Recombinant plasmids containing subgenomic fragments of HCV genotype 1a or 1b were constructed for generation of riboprobes by in vitro transcription from bacteriophage T7 or Sp6 promoters. (C) Dot blot assay for assessing the strand specificity of HCV riboprobes. Unlabeled HCV genomic (sense [S]) and replicative-intermediate (antisense [AS]) target RNAs were synthesized by in vitro transcription from recombinant plasmid pE1-1a, pCORE, or pCU (B), treated with DNase I, and blotted onto nylon membranes. Unlabeled target RNAs were hybridized with Dig-labeled riboprobes synthesized from the genomic (S) or antigenomic (AS) strands of the corresponding recombinant DNA templates. For example, rE1-1a (AS), indicates the antigenomic riboprobe corresponding to the HCV genotype 1a E1 gene. (D) Specificity of positive control riboprobes specific for human beta-actin and HPRT sense RNAs. (E) Specificity of negative control riboprobe for Neo. Unlabeled sense (S) RNA probes and Dig-labeled antisense (AS) RNA probes were generated from recombinant DNA templates containing human and bacterial genes and were hybridized in the dot blot assay.

    Techniques Used: Labeling, Recombinant, Construct, In Vitro, Dot Blot, Synthesized, Plasmid Preparation, Positive Control, Negative Control, Generated

    Generation and analysis of human cell lines expressing HCV genomic and antigenomic RNAs. (A) Construction of expression plasmids pCE-AS and pCE-S. The entire core and E1 genes of HCV genotype 1a were aligned in either the sense orientation (pCE-S) or the antisense orientation (pCE-AS) relative to the human cytomegalovirus major immediate-early promoter regulatory region (P cmv ). Huh7 cells were transfected with plasmid pCE-S or pCE-AS, and stable transformants, designated Huh7-hcvS and Huh7-hcvAS, were selected as described in Materials and Methods. (B) Analysis of HCV RNA expression in cell lines Huh7-hcvS and Huh7-hcvAS by RT-PCR. Total RNA was extracted from cells, digested with DNase I prior to reverse transcription in the presence of a mixture of hexamer (pdN6 ) oligonucleotides (lanes 3 and 5), and then amplified with primers c256 and c186 . The arrow on the right denotes the appropriate expected band of 240 bp. In lanes 4 and 6, the reverse transcription step was eliminated prior to PCR amplification under conditions identical to those used for lanes 3 and 5, respectively. In lanes 1 and 2, RNA from negative control cell lines Huh7 and Huh7-PC3 was analyzed by the same method as in lanes 3 and 5. Lane M contains size markers. (C) Northern dot blot analysis of RNA from transfected or control cell lines with strand-specific HCV riboprobes. CU (S) designates a genomic-strand synthetic HCV RNA which was derived from the pCU clone and which served as a positive control for the antisense riboprobes. See the legend to Fig. for an explanation of other designations.
    Figure Legend Snippet: Generation and analysis of human cell lines expressing HCV genomic and antigenomic RNAs. (A) Construction of expression plasmids pCE-AS and pCE-S. The entire core and E1 genes of HCV genotype 1a were aligned in either the sense orientation (pCE-S) or the antisense orientation (pCE-AS) relative to the human cytomegalovirus major immediate-early promoter regulatory region (P cmv ). Huh7 cells were transfected with plasmid pCE-S or pCE-AS, and stable transformants, designated Huh7-hcvS and Huh7-hcvAS, were selected as described in Materials and Methods. (B) Analysis of HCV RNA expression in cell lines Huh7-hcvS and Huh7-hcvAS by RT-PCR. Total RNA was extracted from cells, digested with DNase I prior to reverse transcription in the presence of a mixture of hexamer (pdN6 ) oligonucleotides (lanes 3 and 5), and then amplified with primers c256 and c186 . The arrow on the right denotes the appropriate expected band of 240 bp. In lanes 4 and 6, the reverse transcription step was eliminated prior to PCR amplification under conditions identical to those used for lanes 3 and 5, respectively. In lanes 1 and 2, RNA from negative control cell lines Huh7 and Huh7-PC3 was analyzed by the same method as in lanes 3 and 5. Lane M contains size markers. (C) Northern dot blot analysis of RNA from transfected or control cell lines with strand-specific HCV riboprobes. CU (S) designates a genomic-strand synthetic HCV RNA which was derived from the pCU clone and which served as a positive control for the antisense riboprobes. See the legend to Fig. for an explanation of other designations.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Negative Control, Northern Blot, Dot Blot, Derivative Assay, Positive Control

    15) Product Images from "Mechanism of Strand-Specific Smooth Muscle ?-Actin Enhancer Interaction by Purine-Rich Element Binding Protein B (Pur?)"

    Article Title: Mechanism of Strand-Specific Smooth Muscle ?-Actin Enhancer Interaction by Purine-Rich Element Binding Protein B (Pur?)

    Journal:

    doi: 10.1021/bi900708j

    Titration analysis of N-HisPurβ binding to SMP382-F by quantitative DNase I footprinting. Representative footprint titration analysis of N-HisPurβ binding to SMP382-F* shows two regions of protection adjacent to the core MCAT motif and
    Figure Legend Snippet: Titration analysis of N-HisPurβ binding to SMP382-F by quantitative DNase I footprinting. Representative footprint titration analysis of N-HisPurβ binding to SMP382-F* shows two regions of protection adjacent to the core MCAT motif and

    Techniques Used: Titration, Binding Assay, Footprinting

    Delineation of a cooperative binding model describing the interaction of N-HisPurβ with the MCAT-containing enhancer based on DNase I footprint data. Individual 3′ and 5′ site data points implying differential N-HisPurβ
    Figure Legend Snippet: Delineation of a cooperative binding model describing the interaction of N-HisPurβ with the MCAT-containing enhancer based on DNase I footprint data. Individual 3′ and 5′ site data points implying differential N-HisPurβ

    Techniques Used: Binding Assay

    16) Product Images from "Neutrophil Extracellular Traps Promote Inflammatory Responses in Psoriasis via Activating Epidermal TLR4/IL-36R Crosstalk"

    Article Title: Neutrophil Extracellular Traps Promote Inflammatory Responses in Psoriasis via Activating Epidermal TLR4/IL-36R Crosstalk

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00746

    Targeting NETs attenuates psoriasis-like inflammation  in vivo (A)  The phenotype and H  E staining of back skin from CI-amidine, DNase I, or PBS-administrated mice with IMQ or Vaseline treatment, three mice per group. Scale bars, 100 μm. ( B)  Quantification of epidermal thickening and infiltrated immunocytes according to H  E staining in a One-way ANOVA,  n  = 8 per group (mean±SD).  (C)  QRT-PCR analyses of psoriasis-related cytokines and molecules from skin samples of mice described as in A One-way ANOVA,  n  = 8 per group (mean±SD).  (D, E)  Representative immunofluorescence and quantification of skin sections from CI-amidine, DNase I, or PBS-treated IMQ mice for neutrophil marker Ly-6G and T cell marker CD4. Scale bars, 100 μm. One-way ANOVA,  n  = 8 per group (mean±SD).  (F)  The MPO-DNA complex quantification indicating NETs level in serum from psoriasis patients and normal controls.  n  = 8, each data point represents an individual. One-way ANOVA. * P
    Figure Legend Snippet: Targeting NETs attenuates psoriasis-like inflammation in vivo (A) The phenotype and H E staining of back skin from CI-amidine, DNase I, or PBS-administrated mice with IMQ or Vaseline treatment, three mice per group. Scale bars, 100 μm. ( B) Quantification of epidermal thickening and infiltrated immunocytes according to H E staining in a One-way ANOVA, n = 8 per group (mean±SD). (C) QRT-PCR analyses of psoriasis-related cytokines and molecules from skin samples of mice described as in A One-way ANOVA, n = 8 per group (mean±SD). (D, E) Representative immunofluorescence and quantification of skin sections from CI-amidine, DNase I, or PBS-treated IMQ mice for neutrophil marker Ly-6G and T cell marker CD4. Scale bars, 100 μm. One-way ANOVA, n = 8 per group (mean±SD). (F) The MPO-DNA complex quantification indicating NETs level in serum from psoriasis patients and normal controls. n = 8, each data point represents an individual. One-way ANOVA. * P

    Techniques Used: In Vivo, Staining, Mouse Assay, Quantitative RT-PCR, Immunofluorescence, Marker

    17) Product Images from "Long-term culture and significant expansion of human Sertoli cells whilst maintaining stable global phenotype and AKT and SMAD1/5 activation"

    Article Title: Long-term culture and significant expansion of human Sertoli cells whilst maintaining stable global phenotype and AKT and SMAD1/5 activation

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-015-0101-2

    Morphology of the testes from OA patients and the isolation of human Sertoli cells from patients with OA. (A)  H  E staining revealed the morphology of testicular tissues from OA patients.  (B)  Seminiferous tubules were obtained after the first enzymatic digestion with collagenase IV and DNase I.  (C)  The mixture of male germ cells and Sertoli cells was isolated from seminiferous tubules using the second enzymatic digestion.  (D)  Sertoli cells remained in the culture plates after differential plating with the removal of male germ cells. Scale bars in  A-C  = 100 μm; scale bar in  D  = 50 μm.
    Figure Legend Snippet: Morphology of the testes from OA patients and the isolation of human Sertoli cells from patients with OA. (A) H E staining revealed the morphology of testicular tissues from OA patients. (B) Seminiferous tubules were obtained after the first enzymatic digestion with collagenase IV and DNase I. (C) The mixture of male germ cells and Sertoli cells was isolated from seminiferous tubules using the second enzymatic digestion. (D) Sertoli cells remained in the culture plates after differential plating with the removal of male germ cells. Scale bars in A-C  = 100 μm; scale bar in D  = 50 μm.

    Techniques Used: Isolation, Staining

    18) Product Images from "Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid"

    Article Title: Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

    Journal: Arthritis & Rheumatology (Hoboken, N.j.)

    doi: 10.1002/art.39313

    Release of peptidyl arginine deiminases (PADs) into the supernatant during NETosis, and detection of enzymatically active PADs.  A,  DNA release in peripheral blood neutrophils from healthy donors that were left unstimulated or stimulated with 25 m M  phorbol myristate acetate (PMA) was assessed for up to 240 minutes after stimulation. Bars show the mean ± SD of 8 samples per group.  B,  For isolation of neutrophil extracellular traps, supernatants (SN) of unstimulated (unst.) or stimulated (stim.) neutrophils were collected. Subsequently, the cells were washed 3 times with RPMI medium (W1–W3), and stimulated cells were incubated in the absence or presence of DNase I; unstimulated cells treated with DNase I served as a control. Bars show the mean ± SD DNA concentration (conc) in 7 samples per group.  C,  Proteins (PAD2, PAD4, and neutrophil elastase [NE]) were precipitated from the same supernatants of unstimulated or stimulated cells as described in  B  and analyzed by Western blotting. Citrullinated proteins (citr.prot.) were detected using chemical modification and anti–modified citrulline antibody. PAD2 and PAD4 antibodies were tested for cross‐reactivity using human (hu) recombinant PAD4 (rPAD4) (250 ng) and human skeletal muscle tissue lysate (15 μg). One representative blot of 4 independent experiments is shown for each group.  D,  PAD activity in supernatants of unstimulated and stimulated cells was compared. Symbols represent individual donors (n = 10); bars show the median.  ∗  =  P
    Figure Legend Snippet: Release of peptidyl arginine deiminases (PADs) into the supernatant during NETosis, and detection of enzymatically active PADs. A, DNA release in peripheral blood neutrophils from healthy donors that were left unstimulated or stimulated with 25 m M phorbol myristate acetate (PMA) was assessed for up to 240 minutes after stimulation. Bars show the mean ± SD of 8 samples per group. B, For isolation of neutrophil extracellular traps, supernatants (SN) of unstimulated (unst.) or stimulated (stim.) neutrophils were collected. Subsequently, the cells were washed 3 times with RPMI medium (W1–W3), and stimulated cells were incubated in the absence or presence of DNase I; unstimulated cells treated with DNase I served as a control. Bars show the mean ± SD DNA concentration (conc) in 7 samples per group. C, Proteins (PAD2, PAD4, and neutrophil elastase [NE]) were precipitated from the same supernatants of unstimulated or stimulated cells as described in B and analyzed by Western blotting. Citrullinated proteins (citr.prot.) were detected using chemical modification and anti–modified citrulline antibody. PAD2 and PAD4 antibodies were tested for cross‐reactivity using human (hu) recombinant PAD4 (rPAD4) (250 ng) and human skeletal muscle tissue lysate (15 μg). One representative blot of 4 independent experiments is shown for each group. D, PAD activity in supernatants of unstimulated and stimulated cells was compared. Symbols represent individual donors (n = 10); bars show the median. ∗  =  P

    Techniques Used: Isolation, Incubation, Concentration Assay, Western Blot, Modification, Recombinant, Activity Assay

    19) Product Images from "Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus"

    Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw777

    SarA binds  srn_9340  on its promoter and represses transcription. ( A ) SarA represses  srn_9340  expression. After 5 h of growth, 10 μg of total RNA were obtained from  Staphylococcus aureus  HG003 and HG003Δ sarA . Srn_9340 expression levels were monitored by northern blot. ( B ) Schematic representation of both the 5′ and 3′ ends of the Srn_9340 transcripts (long and short small RNAs), as determined by rapid amplification of cDNA ends (RACE). ( C ) SarA binds the  srn_9340  promoter  in vitro . An EMSA was done using 10 fmol of a  32 P-labeled  srn_9340  promoter fragment ( srn_9340 P225 ) as a probe in the presence of increasing amounts (0.01–1 pmol) of 6His-tagged SarA.  Srn_9340 P225  forms an initial complex with 0.15 pmol of SarA, and a second one at 0.75 pmol. ( D ) SarA specifically binds  srn_9340 P225 in vitro . EMSA was done with 10 fmol of  32 P-labeled  srn_9340 P225 , 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled  srn_9340 P225 ) or non-specific competitors (unlabeled 16S 225 ). ( E ) DNase I footprinting assays were performed in the presence of 10 fmol  srn_9340 P225 , 7.5.10 −2  U DNase I and increasing amounts (0.1–0.5 pmol) of 6His-SarA. The region protected by SarA is indicated with a vertical dotted arrow, and the numbers indicate relative positions to the previously determined transcription start site (TSS). Lanes G, A, T and C correspond to sequencing. The nucleotides from −51 to −1 ( srn_9340 51 ) are protected by SarA against DNase I degradation. ( F ) Schematic representation of the DNA probes used for EMSA studies. ( G ) Deletion of the protected SarA sequence from the  srn_9340  promoter region abolishes SarA's capacity to bind the  srn_9340  promoter. ( H ) A 51 bp protected sequence is sufficient for SarA binding. EMSA were done using 10 fmol  srn_9340 51  in the presence of increasing amounts of 6His-tagged SarA (0.25–2 pmol). ( I )  Srn_9340 51  competes with  srn_9340 225 for SarA binding. EMSA was performed with 10 fmol of  32 P-labeled  srn_9340 P225 , 0.4 or 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled  srn_9340 51 ) or non-specific competitors (unlabeled  Random 51 ). ( J ) The 23 bp SarA binding site on  srn_9340 225 was confirmed by EMSA done in the presence of 0.5 pmol sarA and with increasing amounts of unlabeled  srn_9340 225 or unlabeled  srn_9340 225 Δ23 .
    Figure Legend Snippet: SarA binds srn_9340 on its promoter and represses transcription. ( A ) SarA represses srn_9340 expression. After 5 h of growth, 10 μg of total RNA were obtained from Staphylococcus aureus HG003 and HG003Δ sarA . Srn_9340 expression levels were monitored by northern blot. ( B ) Schematic representation of both the 5′ and 3′ ends of the Srn_9340 transcripts (long and short small RNAs), as determined by rapid amplification of cDNA ends (RACE). ( C ) SarA binds the srn_9340 promoter in vitro . An EMSA was done using 10 fmol of a 32 P-labeled srn_9340 promoter fragment ( srn_9340 P225 ) as a probe in the presence of increasing amounts (0.01–1 pmol) of 6His-tagged SarA. Srn_9340 P225 forms an initial complex with 0.15 pmol of SarA, and a second one at 0.75 pmol. ( D ) SarA specifically binds srn_9340 P225 in vitro . EMSA was done with 10 fmol of 32 P-labeled srn_9340 P225 , 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340 P225 ) or non-specific competitors (unlabeled 16S 225 ). ( E ) DNase I footprinting assays were performed in the presence of 10 fmol srn_9340 P225 , 7.5.10 −2 U DNase I and increasing amounts (0.1–0.5 pmol) of 6His-SarA. The region protected by SarA is indicated with a vertical dotted arrow, and the numbers indicate relative positions to the previously determined transcription start site (TSS). Lanes G, A, T and C correspond to sequencing. The nucleotides from −51 to −1 ( srn_9340 51 ) are protected by SarA against DNase I degradation. ( F ) Schematic representation of the DNA probes used for EMSA studies. ( G ) Deletion of the protected SarA sequence from the srn_9340 promoter region abolishes SarA's capacity to bind the srn_9340 promoter. ( H ) A 51 bp protected sequence is sufficient for SarA binding. EMSA were done using 10 fmol srn_9340 51 in the presence of increasing amounts of 6His-tagged SarA (0.25–2 pmol). ( I ) Srn_9340 51 competes with srn_9340 225 for SarA binding. EMSA was performed with 10 fmol of 32 P-labeled srn_9340 P225 , 0.4 or 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340 51 ) or non-specific competitors (unlabeled Random 51 ). ( J ) The 23 bp SarA binding site on srn_9340 225 was confirmed by EMSA done in the presence of 0.5 pmol sarA and with increasing amounts of unlabeled srn_9340 225 or unlabeled srn_9340 225 Δ23 .

    Techniques Used: Expressing, Northern Blot, Rapid Amplification of cDNA Ends, In Vitro, Labeling, Footprinting, Sequencing, Binding Assay

    SarA specifically binds a region overlapping the  srn_3610_sprC  promoter and its 5′ end. ( A ) SarA binds the  srn_3610_sprC  promoter  in vitro . Electrophoretic mobility shift assays (EMSA) were done using 10 fmol  sprC P267  probe, a  32 P-labeled  srn_3610_sprC  promoter fragment and increasing amounts of 0.1 to 8 pmol 6His-tagged SarA. ( B ) SarA specifically binds  sprC P267 . EMSA was performed with 4 pmol of SarA and with increasing amounts of specific (unlabeled  sprC P267 ) or non-specific competitors (unlabeled  16S 267 ). The  sprC P267 /SarA complex is only inhibited in the presence of 50–100× excesses of the specific competitor. ( C ) The SarA binding site overlaps the  srn_3610_sprC  promoter and gene. DNase I footprinting assays were performed in the presence of 10 fmol  sprC P267 , 2.10 −2  U DNase I, and increasing amounts (0.5–2 pmol) of 6His-SarA. Lanes 1–4 correspond to  sprC P267  sequencing, and the promoter region is annotated. The vertical dotted arrow indicates the  srn_3610_sprC  region protected from DNase I degradation. The SarA binding site (−32 to +15) was named ‘ sprC 47 .’ ( D ) Schematic representation of the different DNA probes used for EMSA studies. ( E ) The 47 bp region protected by SarA is necessary for SarA binding with  srn_3610_sprC . EMSA were realized as in  A , using  32 P-labeled  sprC P267Δ47  (right) or wild-type  32 P-labeled  sprC P267  (left). SarA was unable to form a complex with  sprC P267Δ47 . ( F ) The 47 bp protected by SarA is sufficient for SarA binding onto  srn_3610_sprC . EMSA were done using 10 fmol  sprC 47 , 4 pmol of 6His-tagged SarA, and increasing amounts of either unlabeled  sprC 47  as a specific competitor or unlabeled 47-bp sequence (Random 47 ) as a non-specific competitor. SarA forms a complex with DNA (up-shifted band) that can only be disrupted by an excess of the specific competitor. ( G ) A 22-bp SarA binding site on  sprC P267  was found with EMSA done in the presence of 0.5 pmol of sarA and with increasing amounts of unlabeled  sprC P267  or unlabeled  sprC P267Δ22 .
    Figure Legend Snippet: SarA specifically binds a region overlapping the srn_3610_sprC promoter and its 5′ end. ( A ) SarA binds the srn_3610_sprC promoter in vitro . Electrophoretic mobility shift assays (EMSA) were done using 10 fmol sprC P267 probe, a 32 P-labeled srn_3610_sprC promoter fragment and increasing amounts of 0.1 to 8 pmol 6His-tagged SarA. ( B ) SarA specifically binds sprC P267 . EMSA was performed with 4 pmol of SarA and with increasing amounts of specific (unlabeled sprC P267 ) or non-specific competitors (unlabeled 16S 267 ). The sprC P267 /SarA complex is only inhibited in the presence of 50–100× excesses of the specific competitor. ( C ) The SarA binding site overlaps the srn_3610_sprC promoter and gene. DNase I footprinting assays were performed in the presence of 10 fmol sprC P267 , 2.10 −2 U DNase I, and increasing amounts (0.5–2 pmol) of 6His-SarA. Lanes 1–4 correspond to sprC P267 sequencing, and the promoter region is annotated. The vertical dotted arrow indicates the srn_3610_sprC region protected from DNase I degradation. The SarA binding site (−32 to +15) was named ‘ sprC 47 .’ ( D ) Schematic representation of the different DNA probes used for EMSA studies. ( E ) The 47 bp region protected by SarA is necessary for SarA binding with srn_3610_sprC . EMSA were realized as in A , using 32 P-labeled sprC P267Δ47 (right) or wild-type 32 P-labeled sprC P267 (left). SarA was unable to form a complex with sprC P267Δ47 . ( F ) The 47 bp protected by SarA is sufficient for SarA binding onto srn_3610_sprC . EMSA were done using 10 fmol sprC 47 , 4 pmol of 6His-tagged SarA, and increasing amounts of either unlabeled sprC 47 as a specific competitor or unlabeled 47-bp sequence (Random 47 ) as a non-specific competitor. SarA forms a complex with DNA (up-shifted band) that can only be disrupted by an excess of the specific competitor. ( G ) A 22-bp SarA binding site on sprC P267 was found with EMSA done in the presence of 0.5 pmol of sarA and with increasing amounts of unlabeled sprC P267 or unlabeled sprC P267Δ22 .

    Techniques Used: In Vitro, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Footprinting, Sequencing

    20) Product Images from "Dynamic metabolic exchange governs a marine algal-bacterial interaction"

    Article Title: Dynamic metabolic exchange governs a marine algal-bacterial interaction

    Journal: eLife

    doi: 10.7554/eLife.17473

    Bacteria induce a unique algal death in aging co-cultures. ( a–d ) Images of cultures demonstrating the change in the culture color over time. ( e–h ) Phase contrast microscopy images. Arrow points to shed coccoliths. ( i–l ) Fluorescent images of chlorophyll and accessory pigments autofluorescence. ( m–p ) Fluorescent images of dead cells stained with Sytox green. Percentages indicate the number of dead cells counted in each population. For each value n  >  300 and the standard deviation between several analyzed fields was up to 20% of the indicated value. ( q–t ) Overlay of phase contrast microscopy images (grey) with fluorescent images of TUNEL assay (green) of cultures at day 20 (see Materials and methods). ( q ) Co-culture, ( r ) Axenic algal culture, ( s ) Positive control, cells were pretreated with DNase I, ( t ) Negative control, the terminal deoxynucleotidyl transferase enzyme (TdT) was replaced with distilled water. Percentages indicate the number of positively stained cells counted in each population. For each value n  >  300 and the standard deviation between several analyzed fields was up to 25% of the indicated value. Scale bar corresponds to 1 µm in e–p and 4 µm in q–t. DOI: http://dx.doi.org/10.7554/eLife.17473.009
    Figure Legend Snippet: Bacteria induce a unique algal death in aging co-cultures. ( a–d ) Images of cultures demonstrating the change in the culture color over time. ( e–h ) Phase contrast microscopy images. Arrow points to shed coccoliths. ( i–l ) Fluorescent images of chlorophyll and accessory pigments autofluorescence. ( m–p ) Fluorescent images of dead cells stained with Sytox green. Percentages indicate the number of dead cells counted in each population. For each value n  >  300 and the standard deviation between several analyzed fields was up to 20% of the indicated value. ( q–t ) Overlay of phase contrast microscopy images (grey) with fluorescent images of TUNEL assay (green) of cultures at day 20 (see Materials and methods). ( q ) Co-culture, ( r ) Axenic algal culture, ( s ) Positive control, cells were pretreated with DNase I, ( t ) Negative control, the terminal deoxynucleotidyl transferase enzyme (TdT) was replaced with distilled water. Percentages indicate the number of positively stained cells counted in each population. For each value n  >  300 and the standard deviation between several analyzed fields was up to 25% of the indicated value. Scale bar corresponds to 1 µm in e–p and 4 µm in q–t. DOI: http://dx.doi.org/10.7554/eLife.17473.009

    Techniques Used: Microscopy, Staining, Standard Deviation, TUNEL Assay, Co-Culture Assay, Positive Control, Negative Control

    Related Articles

    Clone Assay:

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: Paragraph title: Generation of cDNA clones and riboprobes. ... Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Centrifugation:

    Article Title: Pyocyanin Facilitates Extracellular DNA Binding to Pseudomonas aeruginosa Influencing Cell Surface Properties and Aggregation
    Article Snippet: After growth, the P. aeruginosa PA14 strains were harvested and pelleted out by centrifugation at 5000×g for 5 min at 10°C. .. To remove naturally present eDNA from the P. aeruginosa cell surfaces, bacterial suspensions were pre-treated with 40 units (2 units/µl) of DNase I (Life technologies) in the presence of 5 mM MgCl2 for 90 min at room temperature under static conditions and subsequently washed twice with phosphate buffered saline (PBS) and resuspended in PBS.

    Article Title: Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock
    Article Snippet: Escherichia coli ER2267 and HB101 cells harbouring Kpn2I promoter plasmids with or without compatible pCKpn177 plasmid were grown until OD600 = 0.4, cultures we rapidly chilled by adding ice, cells were collected by centrifugation. .. RNA samples were treated with DNase I (Fermentas).

    Amplification:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: A 332-bp promoter region of S. coelicolor amtB was PCR amplified with the primers SCamtBFP(M13F) and SCamtBFP(M13R), and the amplicon was used as the template for further preparation of fluorescent 6-carboxyfluorescein (FAM)-labeled probes with different primer pairs: M13F-FAM and SCamtBFP(M13R) for labeling the coding strand and M13R-FAM and SCamtBFP(M13F) for labeling the noncoding strand. .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: The production of RNA and the subsequent removal of the DNA template were monitored by agarose gel electrophoresis and RNA denaturing gel electrophoresis. .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained. .. DNase I was inactivated by incubation at 65°C for 10 min in the presence of 2.5 mM EDTA.

    Synthesized:

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: Digoxigenin-11-UTP (Dig)-labeled riboprobes were synthesized from purified PCR product templates by runoff transcription with T7 or Sp6 polymerase and with incorporation of Dig according to the manufacturer's protocol (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Electrophoresis:

    Article Title: Tissue specific CTCF occupancy and boundary function at the human growth hormone locus
    Article Snippet: 120 U DNase I (Invitrogen) was then added into the sample and the reaction was incubated at 37°C. .. 120 U DNase I (Invitrogen) was then added into the sample and the reaction was incubated at 37°C.

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet
    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min. .. After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Article Title: An Intronic Locus Control Region Plays an Essential Role in the Establishment of an Autonomous Hepatic Chromatin Domain for the Human Vitamin D-Binding Protein Gene
    Article Snippet: Five hundred micrograms of liver nuclei was suspended in buffer D with 5 mM MgCl2 and incubated on ice for 10 min with increasing amounts of DNase I (Life Technologies). .. Five hundred micrograms of liver nuclei was suspended in buffer D with 5 mM MgCl2 and incubated on ice for 10 min with increasing amounts of DNase I (Life Technologies).

    Incubation:

    Article Title: Pyocyanin Facilitates Extracellular DNA Binding to Pseudomonas aeruginosa Influencing Cell Surface Properties and Aggregation
    Article Snippet: P. aeruginosa PA14 wildtype, DKN370 and Δphz A-G (Note: The DKN370 and Δphz A-G mutants was obtained from Dianne K. Newman) , strains used in this study were plated onto LB agar plates and incubated overnight under aerobic conditions at 37°C. .. To remove naturally present eDNA from the P. aeruginosa cell surfaces, bacterial suspensions were pre-treated with 40 units (2 units/µl) of DNase I (Life technologies) in the presence of 5 mM MgCl2 for 90 min at room temperature under static conditions and subsequently washed twice with phosphate buffered saline (PBS) and resuspended in PBS.

    Article Title: Tissue specific CTCF occupancy and boundary function at the human growth hormone locus
    Article Snippet: Of intact nuclei, 300 μg were suspended in 1 ml RB buffer with 1 mM CaCl2 . .. 120 U DNase I (Invitrogen) was then added into the sample and the reaction was incubated at 37°C. .. At different time points, 50 μg nuclei was removed and the reaction was terminated by adding EDTA to 50 mM followed by digestion of nuclei with an equal volume of solution S (1.6 M NaCl, 1% SDS and 200 μg/ml proteinase K).

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: Following treatment, static cultures were placed back in 37°C, aerobic conditions to complete the 48 h incubation before staining with crystal violet to allow biofilm quantification. .. For restriction enzyme digest of biofilms 4 μl of 10 U/μl Bam HI, Blp I, Hae III, Hin dIII, Msc I or Rsa I, (NEB), or DNase I (Fermentas), or RNase (QIAGEN) were added to test tubes containing diluted C. jejuni suspension prior to static incubation and then incubated at 37°C for 48 h in aerobic conditions. .. Equal volumes (4 μl) of the buffers and bovine serum albumin were also added if recommended by the manufacturers.

    Article Title: Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules
    Article Snippet: A Chirascan CD spectrophotometer (Applied Photophysics, UK) was used to investigate DNA-prodigiosin and DNA-DNase I reactions in a 1-mm path length quartz cuvette. .. For all experiments dsDNA concentration was kept constant (135 ng/μl) with varying prodigiosin concentrations (25, 50, 100, and 500 μM) and DNase I (Invitrogen, USA) (40 units) in 350 μl Milli-Q water were scanned from 200 to 320 nm wavelength range after an incubation for 30-min in a static condition at 25°C. .. Prodigiosin binding to DNA was also assessed using a Varian Cary 100 Bio UV-Visible spectrophotometer (Varian, USA) in a 1-ml quartz cuvette on DNA, prodigiosin, and the DNA-prodigiosin mixture solution in Milli-Q water.

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet
    Article Snippet: Five fmoles of labeled P/T was incubated with 200 fmoles RT and the indicated amounts of dNTP in 10 μl RB buffer for 15 min at 37°C. .. After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min. .. The DNase I digestion was stopped by heating at 90°C for 3 minutes followed by addition of 13 μl loading buffer (16 M urea, 180 mM Tris-HCl, 60 mM taurine, 1 mM EDTA, 0.25% [w/v] bromphenol blue and 0.25% [w/v] xylene cyanol).

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: For both strand assays, 1 pmol (250 ng) of probes was incubated with 3.5-μg mixtures of recombinant GlnR protein and bovine serum albumin in a total volume of 40 μl in the same buffer as for previously described electrophoretic mobility shift assays (EMSAs) ( ). .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C. .. The reaction was stopped by adding 140 μl of DNase I stop solution (200 mM unbuffered sodium acetate, 30 mM EDTA, 0.15% sodium dodecyl sulfate) ( ).

    Article Title: An Intronic Locus Control Region Plays an Essential Role in the Establishment of an Autonomous Hepatic Chromatin Domain for the Human Vitamin D-Binding Protein Gene
    Article Snippet: Nuclei were washed in RB buffer (0.1 M NaCl, 50 mM Tris-HCl [pH 8.0], 3 mM MgCl2 , 0.1 mM PMSF, 5 mM sodium butyrate), pelleted, and resuspended in RB buffer. .. Five hundred micrograms of liver nuclei was suspended in buffer D with 5 mM MgCl2 and incubated on ice for 10 min with increasing amounts of DNase I (Life Technologies). .. EDTA was added to a 25 to 50 mM final concentration to terminate the reactions.

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation
    Article Snippet: Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA. .. Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Approximately 5000 pea root border cells collected as described above were placed in each well of a black 96-well plate (Corning Inc., Corning, NY) with or without 5x106 CFU R . solanacearum . .. Ten units of DNase I (Ambion) or 10 μg of purified NucA or NucB were added along with 2 nM of SYTOX Green DNA stain and reactions were incubated at 25°C for 6 h and fluorescence was measured with a microplate reader at 485nm excitation/528nm emission. .. Relative fluorescence units (RFU) in each well at 6 h were normalized to RFU of the same well at the beginning of the assay.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Overnight cultures of R . solanacearum grown in BMM+0.2% glucose were centrifuged, passed through a 0.2-μm filter, and this cell-free supernatant was incubated with DNase Alert buffer and substrate at 37°C for 3 h according to the manufacturer’s instructions. .. Ten units of DNase I (Ambion, Life Technologies, Carlsbad, CA) and uninoculated BMM were used as positive and negative controls, respectively.

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: Protein (20 μg), binding buffer (as described for the EMSA approach), 2 mM ZnCl2 , 2% polyvinyl alcohol, and 1 μg poly dA-T was incubated for 10 min at room temperature. .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature. .. The DNase I digest was stopped by heating to 75 °C for 10 min.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: The production of RNA and the subsequent removal of the DNA template were monitored by agarose gel electrophoresis and RNA denaturing gel electrophoresis. .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained. .. DNase I was inactivated by incubation at 65°C for 10 min in the presence of 2.5 mM EDTA.

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation
    Article Snippet: Labeled DNA fragments were added to the cell suspension to a final concentration of 1 ng/μl. .. The cells were incubated with DNA at 37°C in 5% CO2 and subsequently treated with 10 U DNase I (recombinant; Fermentas) for another 15 min at 37°C. .. After washing with phosphate-buffered saline, 50 μl of this dilution was applied to clean coverslips for microscopic analysis.

    Article Title: Epstein-Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells
    Article Snippet: In addition, an aliquot of cell lysates was subjected to western blot analysis for the normalization of each transfection efficiency. .. Filtrated (0.45 µm) viral supernatant (160 µl) was incubated with 2 U DNase I (Invitrogen) at 37°C for 30 min followed by extraction of encapsidated EBV DNA using QIAamp MinElute virus spin kit (QIAGEN). .. Each comparative quantitative PCR reaction was composed of 4 µl diluted viral DNA, 5 µl Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA), and 1 µl primer mix (2 µM).

    Gel Extraction:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: After agarose gel electrophoresis, the FAM-labeled probes were purified by using a QIAquick gel extraction kit (Qiagen, Germany) and quantified with a NanoDrop 2000 (Thermo, USA). .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Activity Assay:

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: For restriction enzyme digest of biofilms 4 μl of 10 U/μl Bam HI, Blp I, Hae III, Hin dIII, Msc I or Rsa I, (NEB), or DNase I (Fermentas), or RNase (QIAGEN) were added to test tubes containing diluted C. jejuni suspension prior to static incubation and then incubated at 37°C for 48 h in aerobic conditions. .. For restriction enzyme digest of biofilms 4 μl of 10 U/μl Bam HI, Blp I, Hae III, Hin dIII, Msc I or Rsa I, (NEB), or DNase I (Fermentas), or RNase (QIAGEN) were added to test tubes containing diluted C. jejuni suspension prior to static incubation and then incubated at 37°C for 48 h in aerobic conditions.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Paragraph title: DNase activity assays ... Ten units of DNase I (Ambion, Life Technologies, Carlsbad, CA) and uninoculated BMM were used as positive and negative controls, respectively.

    Article Title: Differential Susceptibility of Germ and Leydig Cells to Cadmium-Mediated Toxicity: Impact on Testis Structure, Adiponectin Levels, and Steroidogenesis
    Article Snippet: Sections were washed in distilled water and incubated with H2 O2 plus methanol (5 min) to stop endogenous peroxidase activity. .. Positive controls were incubated with 1.00 U/mL DNase I (Invitrogen, Waltham, Massachusetts, USA) in DNase buffer for 10 min. Tissue distribution of apoptotic cells was evaluated by computational analysis (Image-Pro Plus, Media Cybernetics, Rockville, Maryland, USA) determining the optical density of brown pixels in all images submitted to the TUNEL assay.

    Spectrophotometry:

    Article Title: Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules
    Article Snippet: A Chirascan CD spectrophotometer (Applied Photophysics, UK) was used to investigate DNA-prodigiosin and DNA-DNase I reactions in a 1-mm path length quartz cuvette. .. For all experiments dsDNA concentration was kept constant (135 ng/μl) with varying prodigiosin concentrations (25, 50, 100, and 500 μM) and DNase I (Invitrogen, USA) (40 units) in 350 μl Milli-Q water were scanned from 200 to 320 nm wavelength range after an incubation for 30-min in a static condition at 25°C.

    Hybridization:

    Article Title: Tissue specific CTCF occupancy and boundary function at the human growth hormone locus
    Article Snippet: 120 U DNase I (Invitrogen) was then added into the sample and the reaction was incubated at 37°C. .. 120 U DNase I (Invitrogen) was then added into the sample and the reaction was incubated at 37°C.

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation
    Article Snippet: The cells were incubated with DNA at 37°C in 5% CO2 and subsequently treated with 10 U DNase I (recombinant; Fermentas) for another 15 min at 37°C. .. Each condition was characterized independently on at least three different days using the same stock of labeled DNA.

    TUNEL Assay:

    Article Title: Differential Susceptibility of Germ and Leydig Cells to Cadmium-Mediated Toxicity: Impact on Testis Structure, Adiponectin Levels, and Steroidogenesis
    Article Snippet: The terminal transferase enzyme was omitted for the negative control. .. Positive controls were incubated with 1.00 U/mL DNase I (Invitrogen, Waltham, Massachusetts, USA) in DNase buffer for 10 min. Tissue distribution of apoptotic cells was evaluated by computational analysis (Image-Pro Plus, Media Cybernetics, Rockville, Maryland, USA) determining the optical density of brown pixels in all images submitted to the TUNEL assay. .. Genomic DNA extraction was carried out according to [ ].

    Gas Chromatography:

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation
    Article Snippet: Several bacterial colonies of 16- to 20-h-old cultures grown on GC agar were resuspended with a 10-μl inoculation syringe in 100 μl DNA uptake medium (GC medium supplemented with IsoVitaleX and 7 mM MgCl2 ) to an optical density at 600 nm (OD600 ) of 0.1. .. The cells were incubated with DNA at 37°C in 5% CO2 and subsequently treated with 10 U DNase I (recombinant; Fermentas) for another 15 min at 37°C.

    Southern Blot:

    Article Title: An Intronic Locus Control Region Plays an Essential Role in the Establishment of an Autonomous Hepatic Chromatin Domain for the Human Vitamin D-Binding Protein Gene
    Article Snippet: Five hundred micrograms of liver nuclei was suspended in buffer D with 5 mM MgCl2 and incubated on ice for 10 min with increasing amounts of DNase I (Life Technologies). .. Five hundred micrograms of liver nuclei was suspended in buffer D with 5 mM MgCl2 and incubated on ice for 10 min with increasing amounts of DNase I (Life Technologies).

    Footprinting:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: Paragraph title: DNase I footprinting assay with FAM-labeled primers. ... After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: Paragraph title: DNase I footprinting ... The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus
    Article Snippet: Paragraph title: DNase I footprinting assay ... Binding reactions were done as previously described, then subjected to 2 or 7.5.10−2 unit of DNase I (Invitrogen) for 1 min at 30°C after supplementing the protein buffer with 40 mM CaCl2 .

    other:

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability
    Article Snippet: Pyocyanin was obtained from Cayman chemicals (Sapphire Biosciences, Sydney, Australia) and DNase I was from Invitrogen (Melbourne, Australia).

    DNA Sequencing:

    Article Title: Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock
    Article Snippet: Paragraph title: RNA extraction, primer extension, and DNA sequencing ... RNA samples were treated with DNase I (Fermentas).

    Polymerase Chain Reaction:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: A 332-bp promoter region of S. coelicolor amtB was PCR amplified with the primers SCamtBFP(M13F) and SCamtBFP(M13R), and the amplicon was used as the template for further preparation of fluorescent 6-carboxyfluorescein (FAM)-labeled probes with different primer pairs: M13F-FAM and SCamtBFP(M13R) for labeling the coding strand and M13R-FAM and SCamtBFP(M13F) for labeling the noncoding strand. .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The PCR conditions were as follows: 1x Titanium Taq PCR buffer, 10 mM ea. dNTPs, 0.4 μM primer, 1x Titanium Taq polymerase (Clontech, US), plasmid containing the wMT-2 promoter in a 50 μl approach; 95 °C 5 min initial denaturation, 95 °C 30 sec, 55 °C 30 sec, 68 °C 30 sec (35 cyles) 68 °C 5 min. .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: Digoxigenin-11-UTP (Dig)-labeled riboprobes were synthesized from purified PCR product templates by runoff transcription with T7 or Sp6 polymerase and with incorporation of Dig according to the manufacturer's protocol (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Recombinant:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: For both strand assays, 1 pmol (250 ng) of probes was incubated with 3.5-μg mixtures of recombinant GlnR protein and bovine serum albumin in a total volume of 40 μl in the same buffer as for previously described electrophoretic mobility shift assays (EMSAs) ( ). .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: PCR products were purified with a PCR purification kit from QIAGEN Inc. (Chatsworth, Calif.) and cloned into plasmid pCRII (Invitrogen, San Diego, Calif.) according to the manufacturer's protocol to generate the recombinant plasmids illustrated in Fig. . .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation
    Article Snippet: Labeled DNA fragments were added to the cell suspension to a final concentration of 1 ng/μl. .. The cells were incubated with DNA at 37°C in 5% CO2 and subsequently treated with 10 U DNase I (recombinant; Fermentas) for another 15 min at 37°C. .. After washing with phosphate-buffered saline, 50 μl of this dilution was applied to clean coverslips for microscopic analysis.

    Nucleic Acid Electrophoresis:

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: The production of RNA and the subsequent removal of the DNA template were monitored by agarose gel electrophoresis and RNA denaturing gel electrophoresis. .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Radioactivity:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet
    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min. .. The samples were reheated for 4 min at 90°C and separated by electrophoresis through a 20% denaturing (8M urea) polyacrylamide gel, at 2000 V for 3-4 h in TTE buffer (90 mM Tris-HCl, 30 mM taurine, 0.5 mM EDTA).

    Fluorescence:

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Approximately 5000 pea root border cells collected as described above were placed in each well of a black 96-well plate (Corning Inc., Corning, NY) with or without 5x106 CFU R . solanacearum . .. Ten units of DNase I (Ambion) or 10 μg of purified NucA or NucB were added along with 2 nM of SYTOX Green DNA stain and reactions were incubated at 25°C for 6 h and fluorescence was measured with a microplate reader at 485nm excitation/528nm emission. .. Relative fluorescence units (RFU) in each well at 6 h were normalized to RFU of the same well at the beginning of the assay.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: DNase activity was read as fluorescence signal released by cleavage of a synthetic oligonucleotide with a fluorescent Hex probe at one end and a fluorescence quencher at the other, using a Synergy HT microplate reader (Biotek Instruments, Winooski, VT). .. Ten units of DNase I (Ambion, Life Technologies, Carlsbad, CA) and uninoculated BMM were used as positive and negative controls, respectively.

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation
    Article Snippet: The cells were incubated with DNA at 37°C in 5% CO2 and subsequently treated with 10 U DNase I (recombinant; Fermentas) for another 15 min at 37°C. .. Each condition was characterized independently on at least three different days using the same stock of labeled DNA.

    RNA Extraction:

    Article Title: Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock
    Article Snippet: Paragraph title: RNA extraction, primer extension, and DNA sequencing ... RNA samples were treated with DNase I (Fermentas).

    Labeling:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet
    Article Snippet: Five fmoles of labeled P/T was incubated with 200 fmoles RT and the indicated amounts of dNTP in 10 μl RB buffer for 15 min at 37°C. .. After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: A 332-bp promoter region of S. coelicolor amtB was PCR amplified with the primers SCamtBFP(M13F) and SCamtBFP(M13R), and the amplicon was used as the template for further preparation of fluorescent 6-carboxyfluorescein (FAM)-labeled probes with different primer pairs: M13F-FAM and SCamtBFP(M13R) for labeling the coding strand and M13R-FAM and SCamtBFP(M13F) for labeling the noncoding strand. .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation
    Article Snippet: Labeled DNA fragments were added to the cell suspension to a final concentration of 1 ng/μl. .. The cells were incubated with DNA at 37°C in 5% CO2 and subsequently treated with 10 U DNase I (recombinant; Fermentas) for another 15 min at 37°C.

    Purification:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: After agarose gel electrophoresis, the FAM-labeled probes were purified by using a QIAquick gel extraction kit (Qiagen, Germany) and quantified with a NanoDrop 2000 (Thermo, USA). .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Approximately 5000 pea root border cells collected as described above were placed in each well of a black 96-well plate (Corning Inc., Corning, NY) with or without 5x106 CFU R . solanacearum . .. Ten units of DNase I (Ambion) or 10 μg of purified NucA or NucB were added along with 2 nM of SYTOX Green DNA stain and reactions were incubated at 25°C for 6 h and fluorescence was measured with a microplate reader at 485nm excitation/528nm emission. .. Relative fluorescence units (RFU) in each well at 6 h were normalized to RFU of the same well at the beginning of the assay.

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature. .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: Digoxigenin-11-UTP (Dig)-labeled riboprobes were synthesized from purified PCR product templates by runoff transcription with T7 or Sp6 polymerase and with incorporation of Dig according to the manufacturer's protocol (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Sequencing:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C. .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock
    Article Snippet: RNA samples were treated with DNase I (Fermentas). .. RNA samples were treated with DNase I (Fermentas).

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature. .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Electrophoretic Mobility Shift Assay:

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: For both strand assays, 1 pmol (250 ng) of probes was incubated with 3.5-μg mixtures of recombinant GlnR protein and bovine serum albumin in a total volume of 40 μl in the same buffer as for previously described electrophoretic mobility shift assays (EMSAs) ( ). .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Lysis:

    Article Title: An Intronic Locus Control Region Plays an Essential Role in the Establishment of an Autonomous Hepatic Chromatin Domain for the Human Vitamin D-Binding Protein Gene
    Article Snippet: Five hundred micrograms of liver nuclei was suspended in buffer D with 5 mM MgCl2 and incubated on ice for 10 min with increasing amounts of DNase I (Life Technologies). .. EDTA was added to a 25 to 50 mM final concentration to terminate the reactions.

    IA:

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: DNase activity was quantified using the DNase Alert Kit (IDT, Coralville, IA). .. Ten units of DNase I (Ambion, Life Technologies, Carlsbad, CA) and uninoculated BMM were used as positive and negative controls, respectively.

    Titration:

    Article Title: Epstein-Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells
    Article Snippet: Paragraph title: Titration of EBV and KSHV viral particles ... Filtrated (0.45 µm) viral supernatant (160 µl) was incubated with 2 U DNase I (Invitrogen) at 37°C for 30 min followed by extraction of encapsidated EBV DNA using QIAamp MinElute virus spin kit (QIAGEN).

    Plasmid Preparation:

    Article Title: Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock
    Article Snippet: Escherichia coli ER2267 and HB101 cells harbouring Kpn2I promoter plasmids with or without compatible pCKpn177 plasmid were grown until OD600 = 0.4, cultures we rapidly chilled by adding ice, cells were collected by centrifugation. .. RNA samples were treated with DNase I (Fermentas).

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The PCR conditions were as follows: 1x Titanium Taq PCR buffer, 10 mM ea. dNTPs, 0.4 μM primer, 1x Titanium Taq polymerase (Clontech, US), plasmid containing the wMT-2 promoter in a 50 μl approach; 95 °C 5 min initial denaturation, 95 °C 30 sec, 55 °C 30 sec, 68 °C 30 sec (35 cyles) 68 °C 5 min. .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained. .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Software:

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature. .. The sample was then purified using PCR purification columns (Qiagen, Netherlands) and eluted in 20 μl elution buffer.

    Negative Control:

    Article Title: Differential Susceptibility of Germ and Leydig Cells to Cadmium-Mediated Toxicity: Impact on Testis Structure, Adiponectin Levels, and Steroidogenesis
    Article Snippet: Positive controls were incubated with 1.00 U/mL DNase I (Invitrogen, Waltham, Massachusetts, USA) in DNase buffer for 10 min. Tissue distribution of apoptotic cells was evaluated by computational analysis (Image-Pro Plus, Media Cybernetics, Rockville, Maryland, USA) determining the optical density of brown pixels in all images submitted to the TUNEL assay. .. Positive controls were incubated with 1.00 U/mL DNase I (Invitrogen, Waltham, Massachusetts, USA) in DNase buffer for 10 min. Tissue distribution of apoptotic cells was evaluated by computational analysis (Image-Pro Plus, Media Cybernetics, Rockville, Maryland, USA) determining the optical density of brown pixels in all images submitted to the TUNEL assay.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained. .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    Binding Assay:

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: Protein (20 μg), binding buffer (as described for the EMSA approach), 2 mM ZnCl2 , 2% polyvinyl alcohol, and 1 μg poly dA-T was incubated for 10 min at room temperature. .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus
    Article Snippet: Klenow Fragment (New England Biolabs) filled the restriction site with [α-32 P] ATP and other unlabeled nucleotides. .. Binding reactions were done as previously described, then subjected to 2 or 7.5.10−2 unit of DNase I (Invitrogen) for 1 min at 30°C after supplementing the protein buffer with 40 mM CaCl2 . .. Reactions were halted by adding a stop buffer (10 μg/ml Invitrogen Proteinase K, 400 mM acetate sodium, 0.2% SDS, 10 Mm EDTA and 50 μg/ml yeast tRNA).

    Agarose Gel Electrophoresis:

    Article Title: Tissue specific CTCF occupancy and boundary function at the human growth hormone locus
    Article Snippet: 120 U DNase I (Invitrogen) was then added into the sample and the reaction was incubated at 37°C. .. 120 U DNase I (Invitrogen) was then added into the sample and the reaction was incubated at 37°C.

    Article Title: Characterization of a New GlnR Binding Box in the Promoter of amtB in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of amtB
    Article Snippet: After agarose gel electrophoresis, the FAM-labeled probes were purified by using a QIAquick gel extraction kit (Qiagen, Germany) and quantified with a NanoDrop 2000 (Thermo, USA). .. After incubation for 30 min at 25°C, 10 μl of a solution containing 0.015 U of DNase I (Invitrogen) and 100 nmol of freshly prepared CaCl2 was added, followed by further incubation for 1 min at 25°C.

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: The production of RNA and the subsequent removal of the DNA template were monitored by agarose gel electrophoresis and RNA denaturing gel electrophoresis. .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained.

    In Vitro:

    Article Title: In Situ Distribution of Hepatitis C Virus Replicative-Intermediate RNA in Hepatic Tissue and Its Correlation with Liver Disease
    Article Snippet: The production of RNA and the subsequent removal of the DNA template were monitored by agarose gel electrophoresis and RNA denaturing gel electrophoresis. .. Briefly, 20 μl of in vitro transcription reaction mixture was incubated with 1 μl of DNase I (amplification grade) (Life Technologies, Gaithersburg, Md.) for 1 h or until the DNA template was invisible on agarose gels while a strong RNA band remained. .. DNase I was inactivated by incubation at 65°C for 10 min in the presence of 2.5 mM EDTA.

    Size-exclusion Chromatography:

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The PCR conditions were as follows: 1x Titanium Taq PCR buffer, 10 mM ea. dNTPs, 0.4 μM primer, 1x Titanium Taq polymerase (Clontech, US), plasmid containing the wMT-2 promoter in a 50 μl approach; 95 °C 5 min initial denaturation, 95 °C 30 sec, 55 °C 30 sec, 68 °C 30 sec (35 cyles) 68 °C 5 min. .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Ethanol Precipitation:

    Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus
    Article Snippet: Binding reactions were done as previously described, then subjected to 2 or 7.5.10−2 unit of DNase I (Invitrogen) for 1 min at 30°C after supplementing the protein buffer with 40 mM CaCl2 . .. Binding reactions were done as previously described, then subjected to 2 or 7.5.10−2 unit of DNase I (Invitrogen) for 1 min at 30°C after supplementing the protein buffer with 40 mM CaCl2 .

    Distillation:

    Article Title: Inhibition of Heat Shock Transcription Factor Binding by a Linear Polyamide Binding in an Unusual 1:1 Mode
    Article Snippet: DNase I was from Invitrogen. .. DNase I was from Invitrogen.

    Apoptosis Assay:

    Article Title: Differential Susceptibility of Germ and Leydig Cells to Cadmium-Mediated Toxicity: Impact on Testis Structure, Adiponectin Levels, and Steroidogenesis
    Article Snippet: Paragraph title: 2.6. In Situ Apoptosis Assay ... Positive controls were incubated with 1.00 U/mL DNase I (Invitrogen, Waltham, Massachusetts, USA) in DNase buffer for 10 min. Tissue distribution of apoptotic cells was evaluated by computational analysis (Image-Pro Plus, Media Cybernetics, Rockville, Maryland, USA) determining the optical density of brown pixels in all images submitted to the TUNEL assay.

    Concentration Assay:

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation. .. For restriction enzyme digest of biofilms 4 μl of 10 U/μl Bam HI, Blp I, Hae III, Hin dIII, Msc I or Rsa I, (NEB), or DNase I (Fermentas), or RNase (QIAGEN) were added to test tubes containing diluted C. jejuni suspension prior to static incubation and then incubated at 37°C for 48 h in aerobic conditions.

    Article Title: Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules
    Article Snippet: A Chirascan CD spectrophotometer (Applied Photophysics, UK) was used to investigate DNA-prodigiosin and DNA-DNase I reactions in a 1-mm path length quartz cuvette. .. For all experiments dsDNA concentration was kept constant (135 ng/μl) with varying prodigiosin concentrations (25, 50, 100, and 500 μM) and DNase I (Invitrogen, USA) (40 units) in 350 μl Milli-Q water were scanned from 200 to 320 nm wavelength range after an incubation for 30-min in a static condition at 25°C. .. Prodigiosin binding to DNA was also assessed using a Varian Cary 100 Bio UV-Visible spectrophotometer (Varian, USA) in a 1-ml quartz cuvette on DNA, prodigiosin, and the DNA-prodigiosin mixture solution in Milli-Q water.

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The probe was then gel-purified (gel purification kit from Qiagen, Netherlands) and the DNA concentration was measured using a NanoDrop (Thermo Scientific, USA). .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Article Title: Single-Stranded DNA Uptake during Gonococcal Transformation
    Article Snippet: Labeled DNA fragments were added to the cell suspension to a final concentration of 1 ng/μl. .. The cells were incubated with DNA at 37°C in 5% CO2 and subsequently treated with 10 U DNase I (recombinant; Fermentas) for another 15 min at 37°C.

    Thin Layer Chromatography:

    Article Title: Inhibition of Heat Shock Transcription Factor Binding by a Linear Polyamide Binding in an Unusual 1:1 Mode
    Article Snippet: DNase I was from Invitrogen. .. DNase I was from Invitrogen.

    In Situ:

    Article Title: Differential Susceptibility of Germ and Leydig Cells to Cadmium-Mediated Toxicity: Impact on Testis Structure, Adiponectin Levels, and Steroidogenesis
    Article Snippet: Paragraph title: 2.6. In Situ Apoptosis Assay ... Positive controls were incubated with 1.00 U/mL DNase I (Invitrogen, Waltham, Massachusetts, USA) in DNase buffer for 10 min. Tissue distribution of apoptotic cells was evaluated by computational analysis (Image-Pro Plus, Media Cybernetics, Rockville, Maryland, USA) determining the optical density of brown pixels in all images submitted to the TUNEL assay.

    Gel Purification:

    Article Title: Metallothionein gene activation in the earthworm (Lumbricus rubellus)
    Article Snippet: The probe was then gel-purified (gel purification kit from Qiagen, Netherlands) and the DNA concentration was measured using a NanoDrop (Thermo Scientific, USA). .. The probe (500 ng) was then added, brought to a total volume of 30 μl and left on ice for 30 min. DNase I (0.1 U) (Life Technologies, USA), 10x buffer and 20 μl of distilled water were added and incubated for 5 min at room temperature.

    Staining:

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: Following treatment, static cultures were placed back in 37°C, aerobic conditions to complete the 48 h incubation before staining with crystal violet to allow biofilm quantification. .. For restriction enzyme digest of biofilms 4 μl of 10 U/μl Bam HI, Blp I, Hae III, Hin dIII, Msc I or Rsa I, (NEB), or DNase I (Fermentas), or RNase (QIAGEN) were added to test tubes containing diluted C. jejuni suspension prior to static incubation and then incubated at 37°C for 48 h in aerobic conditions.

    Article Title: Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum
    Article Snippet: Approximately 5000 pea root border cells collected as described above were placed in each well of a black 96-well plate (Corning Inc., Corning, NY) with or without 5x106 CFU R . solanacearum . .. Ten units of DNase I (Ambion) or 10 μg of purified NucA or NucB were added along with 2 nM of SYTOX Green DNA stain and reactions were incubated at 25°C for 6 h and fluorescence was measured with a microplate reader at 485nm excitation/528nm emission. .. Relative fluorescence units (RFU) in each well at 6 h were normalized to RFU of the same well at the beginning of the assay.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher rnase free
    alTERC gene is transcribed to RNA in vivo in mouse organs and in vitro in mouse cell lines A. RNA was extracted from adult mouse brain, n = 3, cDNA was generated and subjected to PCR analysis using the set 1 primers for mTERC and set 2 primers for alTERC. Two bands of ~220 bp for mTERC and ~150bp for alTERC were observed. NTC- control, no cDNA. (A is a representative picture of 3 independent experiments). B. The PCR reaction described in A was carried out in the presence of radioactive nucleotide (dCTP [αp 32 ] and the reaction products were analysed by 14% polyacrylamide gel electrophoresis following autoradiography. Two bands of ~230 bp and ~210 bp were observed with set 1 primers (Fpr1) for mTERC and one band of ~150 bp with set primers 2 (Fpr2) for alTERC were detected. C. RNA was extracted from mouse NSC-34 motor neurons like cells followed by cDNA production in the presence or absence of <t>DNase</t> or <t>RNase</t> and subjected to PCR amplification as described in A using the set1 and set 2 primers. D. RNA was extracted from mouse organs (brain and spleen) or from mouse neuroblastoma cell line (N2a) and subjected to sqPCR analysis using the set 1 and 2 primers for TERC and alTERC and GAPDH primers as control. A Representative picture of 3 independent experiments. E. The results of experiments described in D were quantified by densitometric analysis with the EZQuant software, calculated relatively to GAPDH and the alTERC/TERC expression ratio was estimated. The data are means ± SD of 3 independent experiments.
    Rnase Free, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase free/product/Thermo Fisher
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    rnase free - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    77
    Thermo Fisher dnase i
    Footprint analysis of IS 1535  deletion substrate LE(v54-20v) containing the minimal transposon sequences required for efficient PEC assembly. ( A ) DNase I footprints of PEC assembly reactions on 5′- 32 P-labeled bottom and top strands of LE(v54-20v). TnpA concentrations were from 4 to 128 nM in 2-fold increasing amounts. Shaded rectangles on the left of the gels denote the positions of transposon sequences; coordinates labeled with v are vector sequences with vH being the equivalent locations of host DNA. The bars on the right of the gels denote regions of significant changes in DNase I reactivity by TnpA with dashes indicating weakly protected regions. ( B ) Exonuclease III delineated boundaries of TnpA binding. TnpA concentrations are the same as in panel A. ( C ) Summary of DNase I (strongly protected regions, blue) and Exo III digestion boundaries on the LE(v54-20v) sequence. Small letters denote vector sequence.
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Thermo Fisher
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-01
    77/100 stars
      Buy from Supplier

    93
    Thermo Fisher deoxyribonuclease i alexa fluor 488 conjugate
    Effects of SSi6 and 6G on induction of autophagy (A) MDA-MB-231 and MCF-10A cells were treated with indicated concentrations of SSi6 and 6G for 6h. Rapamycin 500nM for 24h was used as a positive control of autophagy. Cells were fixed, stained with acridine orange (AO) and images were captured with ImageXpress micro, detecting the formation of acid vesicular organelles (AVOs). Nuclei were stained with DAPI, scale bar = 50 μm. (B) MDA-MB-231 and MCF-10A were treated with SSi6 (50μM) and 6G (100μM) for 6h. Cells were incubated with anti-LC3B and <t>Alexa</t> fluor 488 ® . Wortmannin was used as an inhibitor of autophagy, previously to the treatment with SSi6 (Wort 30μM + 50μM of SSi6). Images were obtained with amplification of 400×. White arrows indicate the labeling of the LC3B protein after incubation of rapamycin and SSi6 for 24 and 6h, respectively.
    Deoxyribonuclease I Alexa Fluor 488 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxyribonuclease i alexa fluor 488 conjugate/product/Thermo Fisher
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    deoxyribonuclease i alexa fluor 488 conjugate - by Bioz Stars, 2020-01
    93/100 stars
      Buy from Supplier

    Image Search Results


    alTERC gene is transcribed to RNA in vivo in mouse organs and in vitro in mouse cell lines A. RNA was extracted from adult mouse brain, n = 3, cDNA was generated and subjected to PCR analysis using the set 1 primers for mTERC and set 2 primers for alTERC. Two bands of ~220 bp for mTERC and ~150bp for alTERC were observed. NTC- control, no cDNA. (A is a representative picture of 3 independent experiments). B. The PCR reaction described in A was carried out in the presence of radioactive nucleotide (dCTP [αp 32 ] and the reaction products were analysed by 14% polyacrylamide gel electrophoresis following autoradiography. Two bands of ~230 bp and ~210 bp were observed with set 1 primers (Fpr1) for mTERC and one band of ~150 bp with set primers 2 (Fpr2) for alTERC were detected. C. RNA was extracted from mouse NSC-34 motor neurons like cells followed by cDNA production in the presence or absence of DNase or RNase and subjected to PCR amplification as described in A using the set1 and set 2 primers. D. RNA was extracted from mouse organs (brain and spleen) or from mouse neuroblastoma cell line (N2a) and subjected to sqPCR analysis using the set 1 and 2 primers for TERC and alTERC and GAPDH primers as control. A Representative picture of 3 independent experiments. E. The results of experiments described in D were quantified by densitometric analysis with the EZQuant software, calculated relatively to GAPDH and the alTERC/TERC expression ratio was estimated. The data are means ± SD of 3 independent experiments.

    Journal: Oncotarget

    Article Title: Expression of functional alternative telomerase RNA component gene in mouse brain and in motor neurons cells protects from oxidative stress

    doi: 10.18632/oncotarget.13049

    Figure Lengend Snippet: alTERC gene is transcribed to RNA in vivo in mouse organs and in vitro in mouse cell lines A. RNA was extracted from adult mouse brain, n = 3, cDNA was generated and subjected to PCR analysis using the set 1 primers for mTERC and set 2 primers for alTERC. Two bands of ~220 bp for mTERC and ~150bp for alTERC were observed. NTC- control, no cDNA. (A is a representative picture of 3 independent experiments). B. The PCR reaction described in A was carried out in the presence of radioactive nucleotide (dCTP [αp 32 ] and the reaction products were analysed by 14% polyacrylamide gel electrophoresis following autoradiography. Two bands of ~230 bp and ~210 bp were observed with set 1 primers (Fpr1) for mTERC and one band of ~150 bp with set primers 2 (Fpr2) for alTERC were detected. C. RNA was extracted from mouse NSC-34 motor neurons like cells followed by cDNA production in the presence or absence of DNase or RNase and subjected to PCR amplification as described in A using the set1 and set 2 primers. D. RNA was extracted from mouse organs (brain and spleen) or from mouse neuroblastoma cell line (N2a) and subjected to sqPCR analysis using the set 1 and 2 primers for TERC and alTERC and GAPDH primers as control. A Representative picture of 3 independent experiments. E. The results of experiments described in D were quantified by densitometric analysis with the EZQuant software, calculated relatively to GAPDH and the alTERC/TERC expression ratio was estimated. The data are means ± SD of 3 independent experiments.

    Article Snippet: In both cases DNA residues were removed using the “DNase I, RNase-free” kit (Thermo Fisher Scientific Inc, Pittsburgh PA, USA).

    Techniques: In Vivo, In Vitro, Generated, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Autoradiography, Amplification, Software, Expressing

    Footprint analysis of IS 1535  deletion substrate LE(v54-20v) containing the minimal transposon sequences required for efficient PEC assembly. ( A ) DNase I footprints of PEC assembly reactions on 5′- 32 P-labeled bottom and top strands of LE(v54-20v). TnpA concentrations were from 4 to 128 nM in 2-fold increasing amounts. Shaded rectangles on the left of the gels denote the positions of transposon sequences; coordinates labeled with v are vector sequences with vH being the equivalent locations of host DNA. The bars on the right of the gels denote regions of significant changes in DNase I reactivity by TnpA with dashes indicating weakly protected regions. ( B ) Exonuclease III delineated boundaries of TnpA binding. TnpA concentrations are the same as in panel A. ( C ) Summary of DNase I (strongly protected regions, blue) and Exo III digestion boundaries on the LE(v54-20v) sequence. Small letters denote vector sequence.

    Journal: eLife

    Article Title: Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition

    doi: 10.7554/eLife.39611

    Figure Lengend Snippet: Footprint analysis of IS 1535 deletion substrate LE(v54-20v) containing the minimal transposon sequences required for efficient PEC assembly. ( A ) DNase I footprints of PEC assembly reactions on 5′- 32 P-labeled bottom and top strands of LE(v54-20v). TnpA concentrations were from 4 to 128 nM in 2-fold increasing amounts. Shaded rectangles on the left of the gels denote the positions of transposon sequences; coordinates labeled with v are vector sequences with vH being the equivalent locations of host DNA. The bars on the right of the gels denote regions of significant changes in DNase I reactivity by TnpA with dashes indicating weakly protected regions. ( B ) Exonuclease III delineated boundaries of TnpA binding. TnpA concentrations are the same as in panel A. ( C ) Summary of DNase I (strongly protected regions, blue) and Exo III digestion boundaries on the LE(v54-20v) sequence. Small letters denote vector sequence.

    Article Snippet: After 60 min incubation at 37°C with TnpAIS1535 , DNase I (0.02 u, Thermo Fisher, Waltham, MA) or Exonuclease III (10 u, NEB, Ipswich, MA) was added for 30 s or 5 min, respectively.

    Techniques: Labeling, Plasmid Preparation, Binding Assay, Sequencing

    EMSA of TnpA IS1535  binding in DNase I footprint reactions. 1 µl aliquots of footprint binding reactions just prior to DNase I digestion were added to 20 µl EMSA buffer and applied to a native gel to assess the relative amounts of PECs in the footprint reactions. TnpA IS1535  concentrations were from 1 to 128 nM.

    Journal: eLife

    Article Title: Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition

    doi: 10.7554/eLife.39611

    Figure Lengend Snippet: EMSA of TnpA IS1535 binding in DNase I footprint reactions. 1 µl aliquots of footprint binding reactions just prior to DNase I digestion were added to 20 µl EMSA buffer and applied to a native gel to assess the relative amounts of PECs in the footprint reactions. TnpA IS1535 concentrations were from 1 to 128 nM.

    Article Snippet: After 60 min incubation at 37°C with TnpAIS1535 , DNase I (0.02 u, Thermo Fisher, Waltham, MA) or Exonuclease III (10 u, NEB, Ipswich, MA) was added for 30 s or 5 min, respectively.

    Techniques: Binding Assay

    Effects of SSi6 and 6G on induction of autophagy (A) MDA-MB-231 and MCF-10A cells were treated with indicated concentrations of SSi6 and 6G for 6h. Rapamycin 500nM for 24h was used as a positive control of autophagy. Cells were fixed, stained with acridine orange (AO) and images were captured with ImageXpress micro, detecting the formation of acid vesicular organelles (AVOs). Nuclei were stained with DAPI, scale bar = 50 μm. (B) MDA-MB-231 and MCF-10A were treated with SSi6 (50μM) and 6G (100μM) for 6h. Cells were incubated with anti-LC3B and Alexa fluor 488 ® . Wortmannin was used as an inhibitor of autophagy, previously to the treatment with SSi6 (Wort 30μM + 50μM of SSi6). Images were obtained with amplification of 400×. White arrows indicate the labeling of the LC3B protein after incubation of rapamycin and SSi6 for 24 and 6h, respectively.

    Journal: Oncotarget

    Article Title: Autophagy-dependent apoptosis is triggered by a semi-synthetic [6]-gingerol analogue in triple negative breast cancer cells

    doi: 10.18632/oncotarget.25704

    Figure Lengend Snippet: Effects of SSi6 and 6G on induction of autophagy (A) MDA-MB-231 and MCF-10A cells were treated with indicated concentrations of SSi6 and 6G for 6h. Rapamycin 500nM for 24h was used as a positive control of autophagy. Cells were fixed, stained with acridine orange (AO) and images were captured with ImageXpress micro, detecting the formation of acid vesicular organelles (AVOs). Nuclei were stained with DAPI, scale bar = 50 μm. (B) MDA-MB-231 and MCF-10A were treated with SSi6 (50μM) and 6G (100μM) for 6h. Cells were incubated with anti-LC3B and Alexa fluor 488 ® . Wortmannin was used as an inhibitor of autophagy, previously to the treatment with SSi6 (Wort 30μM + 50μM of SSi6). Images were obtained with amplification of 400×. White arrows indicate the labeling of the LC3B protein after incubation of rapamycin and SSi6 for 24 and 6h, respectively.

    Article Snippet: For immunostaining assay the anti-rabbit Alexa Fluor 488-conjugated antibody (Thermo Scientific, D12371) was used.

    Techniques: Multiple Displacement Amplification, Positive Control, Staining, Incubation, Amplification, Labeling