dnase i buffer  (Thermo Fisher)


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    Name:
    DNase I Buffer 10X
    Description:
    The same buffer provided with our Ambion DNase I AM2222 AM2224 and recombinant DNase I AM2235 enzymes offered here as a stand alone product in case extra buffer is needed
    Catalog Number:
    am8170g
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher dnase i buffer
    Analysis of the Mur34 binding site by <t>DNase</t> 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.
    The same buffer provided with our Ambion DNase I AM2222 AM2224 and recombinant DNase I AM2235 enzymes offered here as a stand alone product in case extra buffer is needed
    https://www.bioz.com/result/dnase i buffer/product/Thermo Fisher
    Average 99 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    dnase i buffer - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) 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

    2) Product Images from "The Neisseria gonorrhoeae Biofilm Matrix Contains DNA, and an Endogenous Nuclease Controls Its Incorporation ▿"

    Article Title: The Neisseria gonorrhoeae Biofilm Matrix Contains DNA, and an Endogenous Nuclease Controls Its Incorporation ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01162-10

    Evidence that the gonococcal nuclease is a thermonuclease whose activity may be modulated by methylation of DNA. (A, B, and C) Effects of the nuclease at different concentrations on 3 μg herring sperm DNA (A and C) or plasmid DNA (B). The nuclease in panel C was boiled for 5 min. In panels A to D, + indicates a control in which DNase I at 1 μg was used to digest the DNA and − indicates a lane in which the DNase I is omitted. (D) Activities of gonococcal nuclease against DNAs from the different microbes listed above the panel. The amount of nuclease (Nuc) in micrograms is listed above each lane. As can be seen, gonococcal DNA was relatively resistant to gonococcal nuclease compared to DNAs from the other microbes. We tested whether unmethylated gonococcal DNA would be susceptible using 1 μg genomic gonococcal DNA and 1 μg DNA from amplification of a 150-bp region of the gonococcal NGO0968 gene. The results of these experiments are seen in panel E, in which lane 1 contains the NGO0968 PCR product (Pp) plus 0.1 μg Nuc, lane 2 contains the NGO0968 Pp with no enzyme, lane 3 contains 1291 gDNA plus 0.05 μg Nuc, lane 4 contains 1291 gDNA with no enzyme, lane 5 contains the NGO0968 Pp plus 0.05 μg Nuc, and lane 6 contains the NGO0968 Pp with no enzyme.
    Figure Legend Snippet: Evidence that the gonococcal nuclease is a thermonuclease whose activity may be modulated by methylation of DNA. (A, B, and C) Effects of the nuclease at different concentrations on 3 μg herring sperm DNA (A and C) or plasmid DNA (B). The nuclease in panel C was boiled for 5 min. In panels A to D, + indicates a control in which DNase I at 1 μg was used to digest the DNA and − indicates a lane in which the DNase I is omitted. (D) Activities of gonococcal nuclease against DNAs from the different microbes listed above the panel. The amount of nuclease (Nuc) in micrograms is listed above each lane. As can be seen, gonococcal DNA was relatively resistant to gonococcal nuclease compared to DNAs from the other microbes. We tested whether unmethylated gonococcal DNA would be susceptible using 1 μg genomic gonococcal DNA and 1 μg DNA from amplification of a 150-bp region of the gonococcal NGO0968 gene. The results of these experiments are seen in panel E, in which lane 1 contains the NGO0968 PCR product (Pp) plus 0.1 μg Nuc, lane 2 contains the NGO0968 Pp with no enzyme, lane 3 contains 1291 gDNA plus 0.05 μg Nuc, lane 4 contains 1291 gDNA with no enzyme, lane 5 contains the NGO0968 Pp plus 0.05 μg Nuc, and lane 6 contains the NGO0968 Pp with no enzyme.

    Techniques Used: Activity Assay, Methylation, Plasmid Preparation, Amplification, Polymerase Chain Reaction

    Effects of DNase I and Nuc on established wild-type 1291 biofilms. Wild-type N. gonorrhoeae 1291 bacteria expressing gfp were grown in continuous-flow chambers for 48 h. Either 2 units/ml of DNase I or 5 μg/ml Nuc was added to the medium flask, and the flow was continued for an additional 5 h. The biofilms were then observed using confocal microscopy, and 4 z series were taken from each of 3 chambers (repeated 3 times). Results of a COMSTAT analysis of the average thickness (left) and total biomass (right) are represented by bar graphs comparing DNase I- and Nuc-treated biofilms with untreated control biofilms. Reductions in the average thicknesses and biomasses of treated biofilms are statistically significant by Student's t test ( P = 0.01).
    Figure Legend Snippet: Effects of DNase I and Nuc on established wild-type 1291 biofilms. Wild-type N. gonorrhoeae 1291 bacteria expressing gfp were grown in continuous-flow chambers for 48 h. Either 2 units/ml of DNase I or 5 μg/ml Nuc was added to the medium flask, and the flow was continued for an additional 5 h. The biofilms were then observed using confocal microscopy, and 4 z series were taken from each of 3 chambers (repeated 3 times). Results of a COMSTAT analysis of the average thickness (left) and total biomass (right) are represented by bar graphs comparing DNase I- and Nuc-treated biofilms with untreated control biofilms. Reductions in the average thicknesses and biomasses of treated biofilms are statistically significant by Student's t test ( P = 0.01).

    Techniques Used: Expressing, Flow Cytometry, Confocal Microscopy

    Enzymatic activities of whole cells measured on cell lysates of N. gonorrhoeae 1291 (▴), 1291 nuc (▪), and 1291 nuc complemented in trans with nuc (○) and on 1 U DNase I (⧫) and the buffer with labeled primer (•). As can be seen, evidence of nuclease activity was present in the lysate from wild-type 1291 and not from the mutant.
    Figure Legend Snippet: Enzymatic activities of whole cells measured on cell lysates of N. gonorrhoeae 1291 (▴), 1291 nuc (▪), and 1291 nuc complemented in trans with nuc (○) and on 1 U DNase I (⧫) and the buffer with labeled primer (•). As can be seen, evidence of nuclease activity was present in the lysate from wild-type 1291 and not from the mutant.

    Techniques Used: Labeling, Activity Assay, Mutagenesis

    3) Product Images from "Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment"

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00699

    DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Figure Legend Snippet: DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Techniques Used: Staining, Standard Deviation, MANN-WHITNEY

    DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Figure Legend Snippet: DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Techniques Used: Incubation, Staining, Concentration Assay, Standard Deviation, MANN-WHITNEY

    Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Figure Legend Snippet: Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Techniques Used: Inhibition, Incubation, Standard Deviation, MANN-WHITNEY

    Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Figure Legend Snippet: Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Techniques Used: Incubation, Standard Deviation, MANN-WHITNEY

    4) Product Images from "Paf1 Has Distinct Roles in Transcription Elongation and Differential Transcript Fate"

    Article Title: Paf1 Has Distinct Roles in Transcription Elongation and Differential Transcript Fate

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2017.01.006

    Mapping Paf1 on Pol2 Using TEF-Seq (A) Schematic showing single or sequential immunoprecipitation of Pol2 (via the components Rpb3 or Rpb2, NET-seq) or TEF:Pol2 complexes (TEF-seq) via epitope tags from cryogenically lysed and DNase I-treated yeast extracts. (B) Examples of Rpb3 and Rpb2 NET-seq and Paf1 TEF-seq profiles on individual genes. Images in integrated genome viewer (IGV) ( Thorvaldsdóttir et al., 2013 ) show loci on the Watson (top, filled arrows) or Crick (bottom, open arrows) strands. Scales allow relative changes to be visualized and are proportional to each other across all IGV images. (C) Comparison of Rpb2-normalized Paf1 metagene profiles from single (blue) or sequential (black) IP procedures aligned at the TSS or PAS (dashed line, right). Pol2-normalized (using either Rpb2 or Rpb3 NET-seq data) TEF metagene profiles are calculated as follows. TEF/Pol2 (TEF-seq counts/NET-seq counts) ratios are calculated for each 10-nt bin across the specified region. For TSS-aligned plots, the final 200 nt of each gene before the TES is excluded. For PAS or TES-aligned plots, the initial 500 nt after the TSS of each gene is excluded. These ratios are multiplied by a TEF and Pol2 subunit-specific scaling factor to enable visualization on the same arbitrary linear scale. The mean (scaling factor × TEF)/Pol2 ratios are then calculated for each 10-nt bin across all genes after first excluding the top 10% and bottom 10% of ratios to prevent single-nucleotide spikes from skewing the mean (see  STAR Methods  for details). See also  Tables S1  and  S2  and  Figures S1  and  S2 .
    Figure Legend Snippet: Mapping Paf1 on Pol2 Using TEF-Seq (A) Schematic showing single or sequential immunoprecipitation of Pol2 (via the components Rpb3 or Rpb2, NET-seq) or TEF:Pol2 complexes (TEF-seq) via epitope tags from cryogenically lysed and DNase I-treated yeast extracts. (B) Examples of Rpb3 and Rpb2 NET-seq and Paf1 TEF-seq profiles on individual genes. Images in integrated genome viewer (IGV) ( Thorvaldsdóttir et al., 2013 ) show loci on the Watson (top, filled arrows) or Crick (bottom, open arrows) strands. Scales allow relative changes to be visualized and are proportional to each other across all IGV images. (C) Comparison of Rpb2-normalized Paf1 metagene profiles from single (blue) or sequential (black) IP procedures aligned at the TSS or PAS (dashed line, right). Pol2-normalized (using either Rpb2 or Rpb3 NET-seq data) TEF metagene profiles are calculated as follows. TEF/Pol2 (TEF-seq counts/NET-seq counts) ratios are calculated for each 10-nt bin across the specified region. For TSS-aligned plots, the final 200 nt of each gene before the TES is excluded. For PAS or TES-aligned plots, the initial 500 nt after the TSS of each gene is excluded. These ratios are multiplied by a TEF and Pol2 subunit-specific scaling factor to enable visualization on the same arbitrary linear scale. The mean (scaling factor × TEF)/Pol2 ratios are then calculated for each 10-nt bin across all genes after first excluding the top 10% and bottom 10% of ratios to prevent single-nucleotide spikes from skewing the mean (see STAR Methods for details). See also Tables S1 and S2 and Figures S1 and S2 .

    Techniques Used: Immunoprecipitation

    5) Product Images from "The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation"

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1397

    A complex composed of Tfam and Polrmt forms at the LSP promoter even in the absence of Tfb2m. ( A ) DNase I footprinting reveals a Tfam footprint (lane 2; solid line marked Tfam) upstream the promoter. Addition of Polrmt and Tfb2m (lane 3) generates additional footprints (solids line marked Polrmt and Polrmt + Tfb2m, respectively), over the promoter and transcription start site as well as upstream the Tfam binding site at position −50 to −60. The dashed line indicates an extended weaker promoter footprint reaching to +20. When only Tfam and Polrmt are added (lane 4), there are still weak footprints over the promoter, and strong footprints over the −50 and −60 region. Also a clear border is visible upstream the −60 footprint ( B ) Δ320-Polrmt leaves a similar, but not identical, footprint at the promoter and the −50 and −60 region in presence of both transcription factors (lane 3) but almost no footprint in absence of Tfb2m (lane 4). ( C ) A schematic representation of the protected areas of the mouse LSP promoter region. Tfam protects around −11 to −40, Polrmt protects the promoter and the transcription start site extending to +20 and two smaller regions around −50 and −60. The Polrmt promoter protection is strongly enhanced by Tfb2m. With the 180° bending effect of Tfam, it becomes clear that the different Polrmt-DNA interaction sites are placed close to each other in space.
    Figure Legend Snippet: A complex composed of Tfam and Polrmt forms at the LSP promoter even in the absence of Tfb2m. ( A ) DNase I footprinting reveals a Tfam footprint (lane 2; solid line marked Tfam) upstream the promoter. Addition of Polrmt and Tfb2m (lane 3) generates additional footprints (solids line marked Polrmt and Polrmt + Tfb2m, respectively), over the promoter and transcription start site as well as upstream the Tfam binding site at position −50 to −60. The dashed line indicates an extended weaker promoter footprint reaching to +20. When only Tfam and Polrmt are added (lane 4), there are still weak footprints over the promoter, and strong footprints over the −50 and −60 region. Also a clear border is visible upstream the −60 footprint ( B ) Δ320-Polrmt leaves a similar, but not identical, footprint at the promoter and the −50 and −60 region in presence of both transcription factors (lane 3) but almost no footprint in absence of Tfb2m (lane 4). ( C ) A schematic representation of the protected areas of the mouse LSP promoter region. Tfam protects around −11 to −40, Polrmt protects the promoter and the transcription start site extending to +20 and two smaller regions around −50 and −60. The Polrmt promoter protection is strongly enhanced by Tfb2m. With the 180° bending effect of Tfam, it becomes clear that the different Polrmt-DNA interaction sites are placed close to each other in space.

    Techniques Used: Footprinting, Binding Assay

    6) 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

    7) Product Images from "Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy"

    Article Title: Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-014-0092-7

    Fluorometric TdT-mediated dUTP nick-end labeling (TUNEL) assay on treated MKN45 cells.  After 48 hours of treatment with bromelain (100 and 200 μg/mL), NAC (5 and 10 mM) or the combination, cells were assayed for TUNEL reactivity and viewed under laser scanning confocal microscope. Green (fluorescein-12-dUTP) and red (propidium iodide) fluorescence correspond to fragmented DNA and the nucleus, respectively, indicating the presence of apoptotic cells in all treatment groups as compared with DNase I-treated cells used as the positive control. Scale bar: 50 μm.
    Figure Legend Snippet: Fluorometric TdT-mediated dUTP nick-end labeling (TUNEL) assay on treated MKN45 cells. After 48 hours of treatment with bromelain (100 and 200 μg/mL), NAC (5 and 10 mM) or the combination, cells were assayed for TUNEL reactivity and viewed under laser scanning confocal microscope. Green (fluorescein-12-dUTP) and red (propidium iodide) fluorescence correspond to fragmented DNA and the nucleus, respectively, indicating the presence of apoptotic cells in all treatment groups as compared with DNase I-treated cells used as the positive control. Scale bar: 50 μm.

    Techniques Used: End Labeling, TUNEL Assay, Microscopy, Fluorescence, Positive Control

    8) Product Images from "Prevention of Biofilm Formation and Removal of Existing Biofilms by Extracellular DNases of Campylobacter jejuni"

    Article Title: Prevention of Biofilm Formation and Removal of Existing Biofilms by Extracellular DNases of Campylobacter jejuni

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121680

    Inactivation of the cje1441 eDNase gene restores biofilm formation by C . jejuni strain RM1221. (A) shows biofilm formation of NCTC 11168 (white bar), Δ 1441 (dark grey bar), RM1221 (black bar) and a Brucella medium only control (light grey bar). The Δ 1441 mutant shows similar levels of biofilm formation to NCTC 11168 and a significant increase in biofilm formation compared to the parent strain RM1221. (B) Shows that the biofilm produced by the Δ 1441 mutant is susceptible to degradation by DNase I (white bar) and leads to levels of staining indistinguishable from the Brucella medium only control (black bars). (C) Shows biofilm formation of the Δ 1441 mutant following secondary co-culture with strain NCTC 11168 (white bars), the Δ 1441 mutant (dark grey bars), Brucella medium (black bars), or the RM1221 parent strain (light grey bars) showing that deletion of cje1441 inhibits the biofilm degrading ability of RM1221. Bars represent the median, error bars show range and significance was measured using Mann-Whitney tests (* = P
    Figure Legend Snippet: Inactivation of the cje1441 eDNase gene restores biofilm formation by C . jejuni strain RM1221. (A) shows biofilm formation of NCTC 11168 (white bar), Δ 1441 (dark grey bar), RM1221 (black bar) and a Brucella medium only control (light grey bar). The Δ 1441 mutant shows similar levels of biofilm formation to NCTC 11168 and a significant increase in biofilm formation compared to the parent strain RM1221. (B) Shows that the biofilm produced by the Δ 1441 mutant is susceptible to degradation by DNase I (white bar) and leads to levels of staining indistinguishable from the Brucella medium only control (black bars). (C) Shows biofilm formation of the Δ 1441 mutant following secondary co-culture with strain NCTC 11168 (white bars), the Δ 1441 mutant (dark grey bars), Brucella medium (black bars), or the RM1221 parent strain (light grey bars) showing that deletion of cje1441 inhibits the biofilm degrading ability of RM1221. Bars represent the median, error bars show range and significance was measured using Mann-Whitney tests (* = P

    Techniques Used: Mutagenesis, Produced, Staining, Co-Culture Assay, MANN-WHITNEY

    9) Product Images from "The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium"

    Article Title: The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium

    Journal: Molecular microbiology

    doi: 10.1111/mmi.13919

    DNaseI Differential DNA Footprint Analysis (DDFA) of the csgB promoter region In vitro DNase I footprinting of the anti-sense strand at the csgB promoter was performed with 130 nM H-NS and 50 nM CsgD, as indicated. Peaks are regions of hypersensitivity, which are suggestive of distorted or bent DNA, whereas valleys indicate sites of protection. Base positions are indicated relative to the TSS. A) H-NS, B) CsgD, or E) CsgD and H-NS were added to the csgB promoter and the fluorescent peak height following DNase I cleavage determined relative to the protein-free control in relative fluorescent units (RFU). The differences compared to the H-NS control (C) and the CsgD control (D) were also analyzed. Data are presented as the mean +/− SD of three replicates.
    Figure Legend Snippet: DNaseI Differential DNA Footprint Analysis (DDFA) of the csgB promoter region In vitro DNase I footprinting of the anti-sense strand at the csgB promoter was performed with 130 nM H-NS and 50 nM CsgD, as indicated. Peaks are regions of hypersensitivity, which are suggestive of distorted or bent DNA, whereas valleys indicate sites of protection. Base positions are indicated relative to the TSS. A) H-NS, B) CsgD, or E) CsgD and H-NS were added to the csgB promoter and the fluorescent peak height following DNase I cleavage determined relative to the protein-free control in relative fluorescent units (RFU). The differences compared to the H-NS control (C) and the CsgD control (D) were also analyzed. Data are presented as the mean +/− SD of three replicates.

    Techniques Used: In Vitro, Footprinting

    10) Product Images from "Epitope mapping using mRNA display and a unidirectional nested deletion library"

    Article Title: Epitope mapping using mRNA display and a unidirectional nested deletion library

    Journal:

    doi: 10.1093/protein/gzi038

    Construction of a unidirectional nested deletion library. ( A ) cDNA library reverse transcribed with dUTP is partially digested with DNase I. A randomly-primed fill-in reaction is performed with degenerate DNA hexamers containing a constant 5’
    Figure Legend Snippet: Construction of a unidirectional nested deletion library. ( A ) cDNA library reverse transcribed with dUTP is partially digested with DNase I. A randomly-primed fill-in reaction is performed with degenerate DNA hexamers containing a constant 5’

    Techniques Used: cDNA Library Assay

    11) Product Images from "TEFM is a potent stimulator of mitochondrial transcription elongation in vitro"

    Article Title: TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv105

    TEFM prevents termination at transcription pause sites and increases POLRMT affinity to DNA. ( A ) In vitro transcription from LSP on the 3000 nts run-off template (Figure 3A ) at different time points (0, 3, 6, 9, 12, 15 and 30 min) in the absence (lanes 1–7) or presence (lanes 8–14) of 40 nM TEFM. The upper part of the figure is underexposed, and the bottom part is overexposed to compensate for a difference in labeling due to transcript length. For comparison, the pre-terminated transcript labeled PT* is shown in both parts. Full figures of both exposures are found in Supplementary Figure S3A and B. CSB region transcripts as well as run-off transcripts are indicated. ( B ) Quantification of run-off transcripts in panel (A). White squares with full black lines indicate samples in the absence of TEFM, and black squares with dotted lines indicate samples in the presence of TEFM. The transcript levels are measured in photostimulated luminescence per area (PSL/mm 2 ) and the time in minutes. ( C ) The same quantification as in panel (B) but for the CSB II transcript. ( D ) Pulse-chase experiment on the 400 nts run-off LSP template. Transcription was initiated in the absence (lanes 1 and 6) or presence of 40 nM of TEFM (lane 11). After 3 min incubation, an excess of cold UTP was added to stop labeling. At this time point (0*), one of the reaction mixtures lacking TEFM was supplemented with 40 nM of TEFM (lanes 6–10). The reactions were then allowed to progress and samples were taken for analysis after 2.5, 5, 10 and 30 min. Transcripts prematurely terminated in the CSB region (PT and CSB II) as well as run-off transcripts are indicated. LMW marker (New England Biolabs) is indicated. ( E ) Microscale thermophoresis on an 18-mer 5′ Alexa488 labeled DNA hybridized to 8 nts of a 12-mer of RNA against POLRMT in the presence of ATP and in the presence or absence of TEFM. In the presence of ATP, the template allows for one nt incorporation before pausing. The estimated fraction bound based on combined thermophoresis and temperature jump data was plotted against POLRMT in the absence, white squares, or in the presence, filled black squares, of 2000 nM TEFM. The K d of the interactions were determined as 47.8 ± 2.88 nM in the absence and 11.2 ± 1.00 nM in the presence of TEFM. The experiments were performed in triplicate and error bars show standard deviation. ( F ) DNase I footprinting on an LSP PCR template. Lane 1 is a no-protein control whereas lane 2 contains the initiation machinery (POLRMT, TFAM and TFB2M) and lane 3 contains the initiation machinery complemented with TEFM. Arrows indicate differences between samples in the absence or presence of TEFM (lanes 2 and 3, respectively). Relative positions to LSP +1 as well as the TFAM binding site are indicated.
    Figure Legend Snippet: TEFM prevents termination at transcription pause sites and increases POLRMT affinity to DNA. ( A ) In vitro transcription from LSP on the 3000 nts run-off template (Figure 3A ) at different time points (0, 3, 6, 9, 12, 15 and 30 min) in the absence (lanes 1–7) or presence (lanes 8–14) of 40 nM TEFM. The upper part of the figure is underexposed, and the bottom part is overexposed to compensate for a difference in labeling due to transcript length. For comparison, the pre-terminated transcript labeled PT* is shown in both parts. Full figures of both exposures are found in Supplementary Figure S3A and B. CSB region transcripts as well as run-off transcripts are indicated. ( B ) Quantification of run-off transcripts in panel (A). White squares with full black lines indicate samples in the absence of TEFM, and black squares with dotted lines indicate samples in the presence of TEFM. The transcript levels are measured in photostimulated luminescence per area (PSL/mm 2 ) and the time in minutes. ( C ) The same quantification as in panel (B) but for the CSB II transcript. ( D ) Pulse-chase experiment on the 400 nts run-off LSP template. Transcription was initiated in the absence (lanes 1 and 6) or presence of 40 nM of TEFM (lane 11). After 3 min incubation, an excess of cold UTP was added to stop labeling. At this time point (0*), one of the reaction mixtures lacking TEFM was supplemented with 40 nM of TEFM (lanes 6–10). The reactions were then allowed to progress and samples were taken for analysis after 2.5, 5, 10 and 30 min. Transcripts prematurely terminated in the CSB region (PT and CSB II) as well as run-off transcripts are indicated. LMW marker (New England Biolabs) is indicated. ( E ) Microscale thermophoresis on an 18-mer 5′ Alexa488 labeled DNA hybridized to 8 nts of a 12-mer of RNA against POLRMT in the presence of ATP and in the presence or absence of TEFM. In the presence of ATP, the template allows for one nt incorporation before pausing. The estimated fraction bound based on combined thermophoresis and temperature jump data was plotted against POLRMT in the absence, white squares, or in the presence, filled black squares, of 2000 nM TEFM. The K d of the interactions were determined as 47.8 ± 2.88 nM in the absence and 11.2 ± 1.00 nM in the presence of TEFM. The experiments were performed in triplicate and error bars show standard deviation. ( F ) DNase I footprinting on an LSP PCR template. Lane 1 is a no-protein control whereas lane 2 contains the initiation machinery (POLRMT, TFAM and TFB2M) and lane 3 contains the initiation machinery complemented with TEFM. Arrows indicate differences between samples in the absence or presence of TEFM (lanes 2 and 3, respectively). Relative positions to LSP +1 as well as the TFAM binding site are indicated.

    Techniques Used: In Vitro, Labeling, Pulse Chase, Incubation, Marker, Microscale Thermophoresis, Standard Deviation, Footprinting, Polymerase Chain Reaction, Binding Assay

    12) Product Images from "Human Mitochondrial Transcription Factor B2 Is Required for Promoter Melting during Initiation of Transcription *"

    Article Title: Human Mitochondrial Transcription Factor B2 Is Required for Promoter Melting during Initiation of Transcription *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.751008

    DNA footprinting reveals that TFB2M is the promoter melting-inducing factor in human mitochondria. A , potassium permanganate footprinting of the LSP region template strand ( TS , lanes 2–5 ) and non-template strand ( NTS , lanes 7–10 ), in the absence or presence of transcription proteins as indicated. Indicated markers are New England Biolabs Low Molecular Weight Marker ( NEB LMW ) and strand-specific GA ladders ( lanes 1 and 6 , Maxam-Gilbert G+A sequencing reaction, see “Experimental Procedures”). The positions on the DNA in relation to the LSP start site ( arrow ) are indicated. B , densitometric scanning profiles of the autoradiographs in A (where background signals, lane 2 for the template strand and lane 7 for the non-template strand, are subtracted) are shown in green for lanes 3 and 8 (TFAM), red for lanes 4 and 9 (TFAM and POLRMT), and blue for lanes 5 and 10 (TFAM, POLRMT and TFB2M). C , DNase I footprinting of the LSP region (template strand labeled) in the absence or presence of transcription proteins as indicated. The positions on the DNA in relation to the LSP start site ( arrow ) are indicated as well as the footprint positions of TFAM and POLRMT/TFB2M. For indicated markers, see the description of panel A . Densitometric scanning profiles of the autoradiographs are shown in black for lane 2 (absence of protein), green for lane 3 (TFAM), red for lane 4 (TFAM and POLRMT), and blue for lane 5 (TFAM, POLRMT and TFB2M). D , the sequential model for transcription initiation with TFB2M induced melting of the promoter.
    Figure Legend Snippet: DNA footprinting reveals that TFB2M is the promoter melting-inducing factor in human mitochondria. A , potassium permanganate footprinting of the LSP region template strand ( TS , lanes 2–5 ) and non-template strand ( NTS , lanes 7–10 ), in the absence or presence of transcription proteins as indicated. Indicated markers are New England Biolabs Low Molecular Weight Marker ( NEB LMW ) and strand-specific GA ladders ( lanes 1 and 6 , Maxam-Gilbert G+A sequencing reaction, see “Experimental Procedures”). The positions on the DNA in relation to the LSP start site ( arrow ) are indicated. B , densitometric scanning profiles of the autoradiographs in A (where background signals, lane 2 for the template strand and lane 7 for the non-template strand, are subtracted) are shown in green for lanes 3 and 8 (TFAM), red for lanes 4 and 9 (TFAM and POLRMT), and blue for lanes 5 and 10 (TFAM, POLRMT and TFB2M). C , DNase I footprinting of the LSP region (template strand labeled) in the absence or presence of transcription proteins as indicated. The positions on the DNA in relation to the LSP start site ( arrow ) are indicated as well as the footprint positions of TFAM and POLRMT/TFB2M. For indicated markers, see the description of panel A . Densitometric scanning profiles of the autoradiographs are shown in black for lane 2 (absence of protein), green for lane 3 (TFAM), red for lane 4 (TFAM and POLRMT), and blue for lane 5 (TFAM, POLRMT and TFB2M). D , the sequential model for transcription initiation with TFB2M induced melting of the promoter.

    Techniques Used: DNA Footprinting, Footprinting, Molecular Weight, Marker, Sequencing, Labeling

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    Reverse Transcription Polymerase Chain Reaction:

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    Real-time Polymerase Chain Reaction:

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    Cell Culture:

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    Generated:

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    Polymerase Chain Reaction:

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    Article Snippet: The viral pellet was resuspended in DNase buffer and split into two aliquots, one of which was treated for 1 h at 37°C with Turbo DNase (Ambion). .. DNA was then isolated by phenol-chloroform purification, and PCR analysis was performed as described above.

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: DNase I footprinting assay The footprinting template was produced by PCR using the primer pair 5′ CAT GCT TGT TAG ACA TAA ATG C (forward) and 5′ CAT GAT TTT GTA AAA TTT TTA CAA GTA C (reversed). .. The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific).

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471
    Article Snippet: For DNase I footprinting assays, PCR reactions were performed in a total volume of 50 µl containing 2 ng template DNA, 10 pmol of each labled primer, 5% DMSO, and 5U sequencing-grade Taq polymerase (Genescript), and DNA fragment of the shorted P-mur33 were purified using the QIAquick PCR Purification Kit (Qiagen). .. For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Recombinant:

    Article Title: Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy
    Article Snippet: Cells were washed twice with ice-cold phosphate buffered saline (PBS), fixed in 4% methanol-free formaldehyde in PBS for 25 minutes at 4°C and permeabilized by 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) in PBS for 5 minutes (fixed cells incubated for 5 minutes with DNase I buffer and treated with 5.5–10 units/mL of DNase I (Ambion, Life Technologies, MA, USA) for 10 minutes were used as positive controls). .. After being washed, cells were covered with 100 μL of Equilibration Buffer for 5-10 minutes at room temperature and treated with 50 μl of recombinant terminal deoxynucleotidyl transferase (rTdT) incubation buffer at 37°C for 60 minutes inside the humidified chamber (cells incubated with an incubation buffer without rTdT enzyme were used as negative controls).

    Article Title: Evolutionary expansion of a regulatory network by counter-silencing
    Article Snippet: The appropriate template was added, as indicated in , to a final concentration of 1 nM, followed by 20 U of recombinant RNasin (Promega, Madison, WI). .. The transcription reaction was incubated for 20 min at room temperature before stopping by the addition of 20 μl of DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 , and 4 U of DNase I (Thermo Fisher Scientific) and incubation for 30 min at 37°C.

    Cellular Antioxidant Activity Assay:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: DNase I footprinting assay The footprinting template was produced by PCR using the primer pair 5′ CAT GCT TGT TAG ACA TAA ATG C (forward) and 5′ CAT GAT TTT GTA AAA TTT TTA CAA GTA C (reversed). .. The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific).

    Isolation:

    Article Title: Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
    Article Snippet: For supernatant viral genomes, virus was isolated by ultracentrifugation over a sorbitol cushion as previously described ( ). .. The viral pellet was resuspended in DNase buffer and split into two aliquots, one of which was treated for 1 h at 37°C with Turbo DNase (Ambion).

    Article Title: A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila
    Article Snippet: Macronuclei were prepared as described previously ( ) except for the elimination of iodoacetamide and butyric acid from nucleus isolation buffer A. HAT activity was quantitatively extracted from macronuclei by extensive digestion with DNase I. .. For DNase I extraction, freshly prepared macronuclei (108 macronuclei/ml) were resuspended in DNase I extraction buffer, which consisted of 25 mM Tris-HCl (pH 8.0), 15 mM NaCl, 10 mM MgCl2 , 0.1 mM CaCl2 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM dithiothreitol (DTT), and 500 U of DNase I (Gibco BRL) per ml, and incubated on ice for 90 min. After extraction, macronuclear remnants and insoluble debris were removed by centrifugation at 50,500 × g for 30 min at 2°C.

    Article Title: The Neisseria gonorrhoeae Biofilm Matrix Contains DNA, and an Endogenous Nuclease Controls Its Incorporation ▿
    Article Snippet: Paragraph title: RNA isolation. ... The nucleic acids were pelleted, washed with 70% ethanol, and dried, and subsequently, the DNA was digested using RNase-free DNase I (NEB; 10 units), 1× DNase I buffer, and 40 units RNaseOUT (Invitrogen) in RNase-free water (100-μl total volume).

    Co-Immunoprecipitation Assay:

    Article Title: PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis
    Article Snippet: .. For the DNase-treated coIP samples, 100 µl of DNase buffer and 20 U DNase (Ambion) were added, and the samples were incubated for 1 h at room temperature. .. The coIP was done by protein A or G beads (Dynabeads; Lifesciences), depending on the antibody source.

    Electrophoretic Mobility Shift Assay:

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471
    Article Snippet: Paragraph title: Electrophoretic mobility shift and DNase I footprinting assays ... For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Purification:

    Article Title: Identification of Host Factors Involved in Human Cytomegalovirus Replication, Assembly, and Egress Using a Two-Step Small Interfering RNA Screen
    Article Snippet: The viral pellet was resuspended in DNase buffer and split into two aliquots, one of which was treated for 1 h at 37°C with Turbo DNase (Ambion). .. DNA was then isolated by phenol-chloroform purification, and PCR analysis was performed as described above.

    Article Title: The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium
    Article Snippet: Purified σS was incubated with RNAP core (New England Biolabs) at a 10:1 ratio, whereas purified σ70 was incubated with RNAP core at a 2:1 ratio. .. The reaction was stopped by adding 20 μL DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 and 4 U of DNase I (Thermo Fisher Scientific), and incubated for 30 min at 37°C.

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471
    Article Snippet: For DNase I footprinting assays, PCR reactions were performed in a total volume of 50 µl containing 2 ng template DNA, 10 pmol of each labled primer, 5% DMSO, and 5U sequencing-grade Taq polymerase (Genescript), and DNA fragment of the shorted P-mur33 were purified using the QIAquick PCR Purification Kit (Qiagen). .. For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Article Title: Epitope mapping using mRNA display and a unidirectional nested deletion library
    Article Snippet: .. After RNase H treatment (Roche) to remove mRNA, the cDNA was purified by spin-column (QIAquick, Qiagen) and randomly digested with DNase I (0.25 U DNase I (Invitrogen) added to 30 pmol cDNA (~1.2 µM final) in ice-cold 1 × DNase I buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2 , and 0.1 mM CaCl2 )) at 15 °C for 10 min. DNase I was removed using DNase Removal Reagent (Ambion). .. A fill-in reaction (Sequenase v2.0, Amersham Biosciences) was performed according to the manufacturer’s instructions with 125 pmol of myc6-N6-FP (5’-ATC TCT GAA GAG GAC CTG NNN NNN) and 200 µM of each dNTP (~0.6 µM cDNA final).

    Sequencing:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific). .. The DNA fragments were recovered with phenol extraction and ethanol precipitation and analysed on 8% denaturing polyacrylamide sequencing gels (1× TBE and 7M urea).

    Article Title: Evolutionary expansion of a regulatory network by counter-silencing
    Article Snippet: The transcription reaction was incubated for 20 min at room temperature before stopping by the addition of 20 μl of DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 , and 4 U of DNase I (Thermo Fisher Scientific) and incubation for 30 min at 37°C. .. IVT reactions were quantified via RT-PCR using strand- and sequence-specific probes to ensure unambiguous quantification of the transcript of interest. cDNA was generated using the QuantiTect RT-PCR kit (Qiagen) following the manufacturer's instructions, with the following modifications: sequence-specific oligonucleotide probes were used as indicated in , instead of the manufacturer's primer mix, and the synthesis step was allowed to proceed for 30 min.

    Article Title: The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium
    Article Snippet: The reaction was stopped by adding 20 μL DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 and 4 U of DNase I (Thermo Fisher Scientific), and incubated for 30 min at 37°C. .. EDTA was added to final concentration of 5 mM and the reactions incubated for 10 min at 65°C to inactivate DNase I. cDNA synthesis was performed as previously ( ) using the sequence-specific reverse oligonucleotide probes as indicated in .

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471
    Article Snippet: For DNase I footprinting assays, PCR reactions were performed in a total volume of 50 µl containing 2 ng template DNA, 10 pmol of each labled primer, 5% DMSO, and 5U sequencing-grade Taq polymerase (Genescript), and DNA fragment of the shorted P-mur33 were purified using the QIAquick PCR Purification Kit (Qiagen). .. For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Labeling:

    Article Title: Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy
    Article Snippet: Paragraph title: TdT-mediated dUTP nick-end labeling assay ... Cells were washed twice with ice-cold phosphate buffered saline (PBS), fixed in 4% methanol-free formaldehyde in PBS for 25 minutes at 4°C and permeabilized by 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) in PBS for 5 minutes (fixed cells incubated for 5 minutes with DNase I buffer and treated with 5.5–10 units/mL of DNase I (Ambion, Life Technologies, MA, USA) for 10 minutes were used as positive controls).

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The reversed primer was labeled with T4 polynucleotide kinase enzyme (New England Biolabs) and γ-32 P ATP (3000 Ci/mmol). .. The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific).

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471
    Article Snippet: 50-fold specific competitor (the same unlabeled specific promoter with the labeled DNA) and 50-fold non-specific competitor (the similar base pair component and length) were separately added into the reaction system, 50-fold poly dI-dC was added into every binding system. .. For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: DNase I footprinting assay The footprinting template was produced by PCR using the primer pair 5′ CAT GCT TGT TAG ACA TAA ATG C (forward) and 5′ CAT GAT TTT GTA AAA TTT TTA CAA GTA C (reversed). .. The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific).

    Mouse Assay:

    Article Title: PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis
    Article Snippet: Coimmunoprecipitation assays Testis material from twenty 14-dpp-old C57BL/6J male mice was extracted in cold phosphate-buffered saline (PBS), homogenized by Dounce homogenizer, passed through a 40-µm cell strainer (352340; Falcon BD), and centrifuged for 5 min at 3000 × g . .. For the DNase-treated coIP samples, 100 µl of DNase buffer and 20 U DNase (Ambion) were added, and the samples were incubated for 1 h at room temperature.

    Plasmid Preparation:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The labelled template was produced using a plasmid (pCR2.1-TOPO) containing the mouse mitochondrial promoter region (mtDNA positions 15942-341) as template. .. The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific).

    Electrophoresis:

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression
    Article Snippet: DNase I enzymatic activity was analyzed in 10% polyacrylamide gels in DNase I reactivation buffer (40 m m Tris pH 7.6, 2 n m CaCl2 , 2 m m Mg Cl2 ) containing 88 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) for 24 h , at 37 °C. .. Sodium dodecyl sulfate was removed after electrophoresis by washing the gel in MilliQ water with 2% Triton X‐100 for 1 h at room temperature.

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression
    Article Snippet: DNase I activity measurements DNase I enzymatic activity was analyzed in 10% polyacrylamide gels in DNase I reactivation buffer (40 mm Tris pH 7.6, 2 nm CaCl2 , 2 mm Mg Cl2 ) containing 88 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) for 24 h , at 37 °C. .. Sodium dodecyl sulfate was removed after electrophoresis by washing the gel in MilliQ water with 2% Triton X‐100 for 1 h at room temperature.

    Negative Control:

    Article Title: PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis
    Article Snippet: For the DNase-treated coIP samples, 100 µl of DNase buffer and 20 U DNase (Ambion) were added, and the samples were incubated for 1 h at room temperature. .. As a negative control, IgG from the same animal species was used.

    Binding Assay:

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471
    Article Snippet: .. For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min. ..

    Agarose Gel Electrophoresis:

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression
    Article Snippet: DNase I enzymatic activity was analyzed in 10% polyacrylamide gels in DNase I reactivation buffer (40 m m Tris pH 7.6, 2 n m CaCl2 , 2 m m Mg Cl2 ) containing 88 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) for 24 h , at 37 °C. .. In addition, a radial diffusion assay was used to measure DNase I activity in 1% agarose gel with 30 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) in the DNase I reaction buffer.

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression
    Article Snippet: DNase I activity measurements DNase I enzymatic activity was analyzed in 10% polyacrylamide gels in DNase I reactivation buffer (40 mm Tris pH 7.6, 2 nm CaCl2 , 2 mm Mg Cl2 ) containing 88 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) for 24 h , at 37 °C. .. In addition, a radial diffusion assay was used to measure DNase I activity in 1% agarose gel with 30 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) in the DNase I reaction buffer.

    Article Title: Epitope mapping using mRNA display and a unidirectional nested deletion library
    Article Snippet: After RNase H treatment (Roche) to remove mRNA, the cDNA was purified by spin-column (QIAquick, Qiagen) and randomly digested with DNase I (0.25 U DNase I (Invitrogen) added to 30 pmol cDNA (~1.2 µM final) in ice-cold 1 × DNase I buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2 , and 0.1 mM CaCl2 )) at 15 °C for 10 min. DNase I was removed using DNase Removal Reagent (Ambion). .. First-strand cDNA was digested with uracil-DNA glycosylase (UDG) and ssDNA > 50 bases was extracted with QiaEX II (Qiagen) from a 4% agarose gel ( ).

    In Vitro:

    Article Title: Evolutionary expansion of a regulatory network by counter-silencing
    Article Snippet: Paragraph title: In vitro transcription ... The transcription reaction was incubated for 20 min at room temperature before stopping by the addition of 20 μl of DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 , and 4 U of DNase I (Thermo Fisher Scientific) and incubation for 30 min at 37°C.

    Article Title: The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium
    Article Snippet: Paragraph title: In vitro transcription ... The reaction was stopped by adding 20 μL DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 and 4 U of DNase I (Thermo Fisher Scientific), and incubated for 30 min at 37°C.

    Ethanol Precipitation:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific). .. The DNA fragments were recovered with phenol extraction and ethanol precipitation and analysed on 8% denaturing polyacrylamide sequencing gels (1× TBE and 7M urea).

    Spectrophotometry:

    Article Title: The Neisseria gonorrhoeae Biofilm Matrix Contains DNA, and an Endogenous Nuclease Controls Its Incorporation ▿
    Article Snippet: The nucleic acids were pelleted, washed with 70% ethanol, and dried, and subsequently, the DNA was digested using RNase-free DNase I (NEB; 10 units), 1× DNase I buffer, and 40 units RNaseOUT (Invitrogen) in RNase-free water (100-μl total volume). .. The RNA was quantitated using a NanoDrop 2000 spectrophotometer.

    Produced:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The labelled template was produced using a plasmid (pCR2.1-TOPO) containing the mouse mitochondrial promoter region (mtDNA positions 15942-341) as template. .. The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific).

    Concentration Assay:

    Article Title: Prevention of Biofilm Formation and Removal of Existing Biofilms by Extracellular DNases of Campylobacter jejuni
    Article Snippet: .. A volume of 4 μl DNase I, to give a final concentration of 4 U/ml (Fermentas) and 4 μl DNase I buffer (Fermentas) was added to test tubes at the start of the incubation. .. Following treatment, biofilms were either re-incubated for the remaining 48 h incubation, washed and crystal violet stained or washed and a new volume of cell culture or Brucella medium added to the tube.

    Article Title: Evolutionary expansion of a regulatory network by counter-silencing
    Article Snippet: Reactions were allowed to equilibrate at room temperature, appropriately diluted H-NS, SlyA, or PhoP were added as indicated, and the reaction incubated for an additional 10 min. For counter-silencing reactions, H-NS was first added and the reaction incubated for 10 min before addition of SlyA or PhoP, after which the reaction was incubated for another 10 min. RNAP holoenzyme (Epicentre, Madison, WI) was then added to a final concentration of 10 nM and the reaction incubated for 10 min before rNTPs were added to a final concentration of 1 mM. .. The transcription reaction was incubated for 20 min at room temperature before stopping by the addition of 20 μl of DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 , and 4 U of DNase I (Thermo Fisher Scientific) and incubation for 30 min at 37°C.

    Article Title: The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium
    Article Snippet: Following the addition of regulatory proteins, reconstituted RNA polymerase holoenyzme was incubated with template for 10 min at a final concentration of 10 nM. rNTPs were added to a final concentration of 1 nM and the reaction incubated for an additional 30 min at 30°C. .. The reaction was stopped by adding 20 μL DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 and 4 U of DNase I (Thermo Fisher Scientific), and incubated for 30 min at 37°C.

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: .. Enzyme treatment of C. jejuni biofilms 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. .. 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.

    HAT Assay:

    Article Title: A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila
    Article Snippet: Macronuclei were prepared as described previously ( ) except for the elimination of iodoacetamide and butyric acid from nucleus isolation buffer A. HAT activity was quantitatively extracted from macronuclei by extensive digestion with DNase I. .. For DNase I extraction, freshly prepared macronuclei (108 macronuclei/ml) were resuspended in DNase I extraction buffer, which consisted of 25 mM Tris-HCl (pH 8.0), 15 mM NaCl, 10 mM MgCl2 , 0.1 mM CaCl2 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM dithiothreitol (DTT), and 500 U of DNase I (Gibco BRL) per ml, and incubated on ice for 90 min. After extraction, macronuclear remnants and insoluble debris were removed by centrifugation at 50,500 × g for 30 min at 2°C.

    CTG Assay:

    Article Title: Epitope mapping using mRNA display and a unidirectional nested deletion library
    Article Snippet: After RNase H treatment (Roche) to remove mRNA, the cDNA was purified by spin-column (QIAquick, Qiagen) and randomly digested with DNase I (0.25 U DNase I (Invitrogen) added to 30 pmol cDNA (~1.2 µM final) in ice-cold 1 × DNase I buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2 , and 0.1 mM CaCl2 )) at 15 °C for 10 min. DNase I was removed using DNase Removal Reagent (Ambion). .. A fill-in reaction (Sequenase v2.0, Amersham Biosciences) was performed according to the manufacturer’s instructions with 125 pmol of myc6-N6-FP (5’-ATC TCT GAA GAG GAC CTG NNN NNN) and 200 µM of each dNTP (~0.6 µM cDNA final).

    Staining:

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression
    Article Snippet: DNase I enzymatic activity was analyzed in 10% polyacrylamide gels in DNase I reactivation buffer (40 m m Tris pH 7.6, 2 n m CaCl2 , 2 m m Mg Cl2 ) containing 88 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) for 24 h , at 37 °C. .. To check for activity, the gel was stained with Nucleic Acid Gel Stain GelRed™ 730–2958 (VWR International, Oslo, Norway), and visualized under UV illuminator.

    Article Title: Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy
    Article Snippet: Cells were washed twice with ice-cold phosphate buffered saline (PBS), fixed in 4% methanol-free formaldehyde in PBS for 25 minutes at 4°C and permeabilized by 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) in PBS for 5 minutes (fixed cells incubated for 5 minutes with DNase I buffer and treated with 5.5–10 units/mL of DNase I (Ambion, Life Technologies, MA, USA) for 10 minutes were used as positive controls). .. Unincorporated fluorescein-12-dUTP was removed by PBS washes and cells were stained with 1 μg/mL propidium iodide in PBS for 15 minutes at room temperature in the dark.

    Article Title: Prevention of Biofilm Formation and Removal of Existing Biofilms by Extracellular DNases of Campylobacter jejuni
    Article Snippet: A volume of 4 μl DNase I, to give a final concentration of 4 U/ml (Fermentas) and 4 μl DNase I buffer (Fermentas) was added to test tubes at the start of the incubation. .. Following treatment, biofilms were either re-incubated for the remaining 48 h incubation, washed and crystal violet stained or washed and a new volume of cell culture or Brucella medium added to the tube.

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression
    Article Snippet: DNase I activity measurements DNase I enzymatic activity was analyzed in 10% polyacrylamide gels in DNase I reactivation buffer (40 mm Tris pH 7.6, 2 nm CaCl2 , 2 mm Mg Cl2 ) containing 88 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) for 24 h , at 37 °C. .. To check for activity, the gel was stained with Nucleic Acid Gel Stain GelRed™ 730–2958 (VWR International, Oslo, Norway), and visualized under UV illuminator.

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment
    Article Snippet: Enzyme treatment of C. jejuni biofilms 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. .. 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.

    Amplification:

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471
    Article Snippet: For the competitive EMSAs, the specific promoter regions of mur33 was amplified by PCR using primers of mur33-PF/mur33-PR11, the promoter DNA was labeled with 5′-FAM synthesized by Sangon Biotech (Shanghai) on the forward primer. .. For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

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    Thermo Fisher dnase i reaction buffer
    Overview of RT-RamDA and single-cell RamDA-seq.  a  Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I.  b  Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog ,  Pou5f1 ,  Zfp42 , and  Sox2 ) and three housekeeping ( Gnb2l1 ,  Atp5a1 , and  Tubb5 ) genes using a conventional method (−) as a standard.  c  Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section.  d  Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in  b  and  d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile.  e  Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in  d  and  e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Dnase I Reaction Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i reaction buffer/product/Thermo Fisher
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    A complex composed of Tfam and Polrmt forms at the LSP promoter even in the absence of Tfb2m. ( A ) <t>DNase</t> I footprinting reveals a Tfam footprint (lane 2; solid line marked Tfam) upstream the promoter. Addition of Polrmt and Tfb2m (lane 3) generates additional footprints (solids line marked Polrmt and Polrmt + Tfb2m, respectively), over the promoter and transcription start site as well as upstream the Tfam binding site at position −50 to −60. The dashed line indicates an extended weaker promoter footprint reaching to +20. When only Tfam and Polrmt are added (lane 4), there are still weak footprints over the promoter, and strong footprints over the −50 and −60 region. Also a clear border is visible upstream the −60 footprint ( B ) Δ320-Polrmt leaves a similar, but not identical, footprint at the promoter and the −50 and −60 region in presence of both transcription factors (lane 3) but almost no footprint in absence of Tfb2m (lane 4). ( C ) A schematic representation of the protected areas of the mouse LSP promoter region. Tfam protects around −11 to −40, Polrmt protects the promoter and the transcription start site extending to +20 and two smaller regions around −50 and −60. The Polrmt promoter protection is strongly enhanced by Tfb2m. With the 180° bending effect of Tfam, it becomes clear that the different Polrmt-DNA interaction sites are placed close to each other in space.
    Dnase I Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i buffer/product/Thermo Fisher
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    dnase i buffer - by Bioz Stars, 2020-04
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    Overview of RT-RamDA and single-cell RamDA-seq.  a  Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I.  b  Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog ,  Pou5f1 ,  Zfp42 , and  Sox2 ) and three housekeeping ( Gnb2l1 ,  Atp5a1 , and  Tubb5 ) genes using a conventional method (−) as a standard.  c  Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section.  d  Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in  b  and  d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile.  e  Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in  d  and  e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    A complex composed of Tfam and Polrmt forms at the LSP promoter even in the absence of Tfb2m. ( A ) DNase I footprinting reveals a Tfam footprint (lane 2; solid line marked Tfam) upstream the promoter. Addition of Polrmt and Tfb2m (lane 3) generates additional footprints (solids line marked Polrmt and Polrmt + Tfb2m, respectively), over the promoter and transcription start site as well as upstream the Tfam binding site at position −50 to −60. The dashed line indicates an extended weaker promoter footprint reaching to +20. When only Tfam and Polrmt are added (lane 4), there are still weak footprints over the promoter, and strong footprints over the −50 and −60 region. Also a clear border is visible upstream the −60 footprint ( B ) Δ320-Polrmt leaves a similar, but not identical, footprint at the promoter and the −50 and −60 region in presence of both transcription factors (lane 3) but almost no footprint in absence of Tfb2m (lane 4). ( C ) A schematic representation of the protected areas of the mouse LSP promoter region. Tfam protects around −11 to −40, Polrmt protects the promoter and the transcription start site extending to +20 and two smaller regions around −50 and −60. The Polrmt promoter protection is strongly enhanced by Tfb2m. With the 180° bending effect of Tfam, it becomes clear that the different Polrmt-DNA interaction sites are placed close to each other in space.

    Journal: Nucleic Acids Research

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation

    doi: 10.1093/nar/gkt1397

    Figure Lengend Snippet: A complex composed of Tfam and Polrmt forms at the LSP promoter even in the absence of Tfb2m. ( A ) DNase I footprinting reveals a Tfam footprint (lane 2; solid line marked Tfam) upstream the promoter. Addition of Polrmt and Tfb2m (lane 3) generates additional footprints (solids line marked Polrmt and Polrmt + Tfb2m, respectively), over the promoter and transcription start site as well as upstream the Tfam binding site at position −50 to −60. The dashed line indicates an extended weaker promoter footprint reaching to +20. When only Tfam and Polrmt are added (lane 4), there are still weak footprints over the promoter, and strong footprints over the −50 and −60 region. Also a clear border is visible upstream the −60 footprint ( B ) Δ320-Polrmt leaves a similar, but not identical, footprint at the promoter and the −50 and −60 region in presence of both transcription factors (lane 3) but almost no footprint in absence of Tfb2m (lane 4). ( C ) A schematic representation of the protected areas of the mouse LSP promoter region. Tfam protects around −11 to −40, Polrmt protects the promoter and the transcription start site extending to +20 and two smaller regions around −50 and −60. The Polrmt promoter protection is strongly enhanced by Tfb2m. With the 180° bending effect of Tfam, it becomes clear that the different Polrmt-DNA interaction sites are placed close to each other in space.

    Article Snippet: The mixture was incubated for 20 min at room temperature followed by addition of 2 μl of 30 mU/μl DNase I diluted in 2.5× DNase I buffer with MgCl2 (Thermo Scientific).

    Techniques: Footprinting, Binding Assay

    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.

    Journal: Nature communications

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

    doi: 10.1038/ncomms6270

    Figure Lengend 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.

    Article Snippet: The transcription reaction was incubated for 20 min at room temperature before stopping by the addition of 20 μl of DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 , and 4 U of DNase I (Thermo Fisher Scientific) and incubation for 30 min at 37°C.

    Techniques: In Vitro, Footprinting, Incubation, Protein Binding

    DNaseI Differential DNA Footprint Analysis (DDFA) of the csgB promoter region In vitro DNase I footprinting of the anti-sense strand at the csgB promoter was performed with 130 nM H-NS and 50 nM CsgD, as indicated. Peaks are regions of hypersensitivity, which are suggestive of distorted or bent DNA, whereas valleys indicate sites of protection. Base positions are indicated relative to the TSS. A) H-NS, B) CsgD, or E) CsgD and H-NS were added to the csgB promoter and the fluorescent peak height following DNase I cleavage determined relative to the protein-free control in relative fluorescent units (RFU). The differences compared to the H-NS control (C) and the CsgD control (D) were also analyzed. Data are presented as the mean +/− SD of three replicates.

    Journal: Molecular microbiology

    Article Title: The curli regulator CsgD mediates stationary phase counter-silencing of csgBA in Salmonella Typhimurium

    doi: 10.1111/mmi.13919

    Figure Lengend Snippet: DNaseI Differential DNA Footprint Analysis (DDFA) of the csgB promoter region In vitro DNase I footprinting of the anti-sense strand at the csgB promoter was performed with 130 nM H-NS and 50 nM CsgD, as indicated. Peaks are regions of hypersensitivity, which are suggestive of distorted or bent DNA, whereas valleys indicate sites of protection. Base positions are indicated relative to the TSS. A) H-NS, B) CsgD, or E) CsgD and H-NS were added to the csgB promoter and the fluorescent peak height following DNase I cleavage determined relative to the protein-free control in relative fluorescent units (RFU). The differences compared to the H-NS control (C) and the CsgD control (D) were also analyzed. Data are presented as the mean +/− SD of three replicates.

    Article Snippet: The reaction was stopped by adding 20 μL DNase I buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2 , 0.1 mM CaCl2 and 4 U of DNase I (Thermo Fisher Scientific), and incubated for 30 min at 37°C.

    Techniques: In Vitro, Footprinting