Structured Review

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RNA chromatin immunoprecipitation (ChIP) assay to determine the interaction of ZIKV protein with viral RNA. a The diagram of ZIKV genome and the area covered by the primers listed in Table 2 : 5′ side, from 1 to 1051; 3′ side, from 10,031 to 10,776. b RNA ChIP assay examined by regular RT-PCR. The Vero cells were infected with ZIKV MR766 at an MOI of 0.5 for 24 h. Then the cells were fixed with 4% formaldehyde. The cells were then sonicated to shear the RNA to 250–500 nt. The samples were then divided into 3 part: 1) input, 2) normal mouse IgG-precipitation, and 3) anti-ZIKV antibody precipitation. In both 2) and 3), the RNA-protein complex was pulled down using an anti-ZIKV mouse serum generated from a ZIKV-infected mouse or the normal mouse IgG (as negative control) with the beads. After washing for 3 cycles, the protein was digested with proteinase K, and the DNA was removed by <t>DNase</t> I <t>(RNase</t> free). The input RNA and the ChIPed RNA samples were then precipitated and the samples were used for RT-PCR. The PCR products were applied to run an agarose gel and visualized. c RNA ChIP assay examined by real-time RT-PCR. The same as B, but used a real-time RT-PCR. The yields of PCR from ChIPed RNA or from input RNA were normalized to IgG. The ratio of PCR products from ChIPed RNA were then compared to the input shown as % Input
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Images

1) Product Images from "Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus"

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2338-4

RNA chromatin immunoprecipitation (ChIP) assay to determine the interaction of ZIKV protein with viral RNA. a The diagram of ZIKV genome and the area covered by the primers listed in Table 2 : 5′ side, from 1 to 1051; 3′ side, from 10,031 to 10,776. b RNA ChIP assay examined by regular RT-PCR. The Vero cells were infected with ZIKV MR766 at an MOI of 0.5 for 24 h. Then the cells were fixed with 4% formaldehyde. The cells were then sonicated to shear the RNA to 250–500 nt. The samples were then divided into 3 part: 1) input, 2) normal mouse IgG-precipitation, and 3) anti-ZIKV antibody precipitation. In both 2) and 3), the RNA-protein complex was pulled down using an anti-ZIKV mouse serum generated from a ZIKV-infected mouse or the normal mouse IgG (as negative control) with the beads. After washing for 3 cycles, the protein was digested with proteinase K, and the DNA was removed by DNase I (RNase free). The input RNA and the ChIPed RNA samples were then precipitated and the samples were used for RT-PCR. The PCR products were applied to run an agarose gel and visualized. c RNA ChIP assay examined by real-time RT-PCR. The same as B, but used a real-time RT-PCR. The yields of PCR from ChIPed RNA or from input RNA were normalized to IgG. The ratio of PCR products from ChIPed RNA were then compared to the input shown as % Input
Figure Legend Snippet: RNA chromatin immunoprecipitation (ChIP) assay to determine the interaction of ZIKV protein with viral RNA. a The diagram of ZIKV genome and the area covered by the primers listed in Table 2 : 5′ side, from 1 to 1051; 3′ side, from 10,031 to 10,776. b RNA ChIP assay examined by regular RT-PCR. The Vero cells were infected with ZIKV MR766 at an MOI of 0.5 for 24 h. Then the cells were fixed with 4% formaldehyde. The cells were then sonicated to shear the RNA to 250–500 nt. The samples were then divided into 3 part: 1) input, 2) normal mouse IgG-precipitation, and 3) anti-ZIKV antibody precipitation. In both 2) and 3), the RNA-protein complex was pulled down using an anti-ZIKV mouse serum generated from a ZIKV-infected mouse or the normal mouse IgG (as negative control) with the beads. After washing for 3 cycles, the protein was digested with proteinase K, and the DNA was removed by DNase I (RNase free). The input RNA and the ChIPed RNA samples were then precipitated and the samples were used for RT-PCR. The PCR products were applied to run an agarose gel and visualized. c RNA ChIP assay examined by real-time RT-PCR. The same as B, but used a real-time RT-PCR. The yields of PCR from ChIPed RNA or from input RNA were normalized to IgG. The ratio of PCR products from ChIPed RNA were then compared to the input shown as % Input

Techniques Used: Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Infection, Sonication, Generated, Negative Control, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR

2) Product Images from "Aflatoxin Biosynthesis Is a Novel Source of Reactive Oxygen Species—A Potential Redox Signal to Initiate Resistance to Oxidative Stress?"

Article Title: Aflatoxin Biosynthesis Is a Novel Source of Reactive Oxygen Species—A Potential Redox Signal to Initiate Resistance to Oxidative Stress?

Journal: Toxins

doi: 10.3390/toxins7051411

Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.
Figure Legend Snippet: Analysis of transcript levels in 7 h germlings. A. parasiticus SU-1, AFS10, and Δ veA were grown on YES solid media and spores were collected at 5 days. Germlings were grown from fresh spores in GMS liquid medium for 7 h, frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from germlings using grinding and sonication as described in Methods. RT-PCR was performed on total RNA treated with RNAse-free DNAse I with primers specific for each gene ( Table 3 ). PCR products were separated on a 1% agarose gel by electrophoresis. Citrate synthase, a constitutively expressed gene, was used as a positive control. gDNA, genomic DNA.

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

3) Product Images from "Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation"

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067647

The T6SS gene cluster consists of the imp and hcp operons transcribed from divergent promoters. ( A ) The imp operon consisting of 14 genes ( atu4343 to atu4330 ) is in filled colors and the hcp operon encoding nine genes ( atu4344 to atu4352 ) and atu3642 ( vgrG-2 ) encoded elsewhere is in open colors. The genes (indicated with locus names) with homology to conserved T6SS components are designated as tss (type VI secretion) or tag (type VI secretion-associated gene) based on nomenclature proposed by Shalom et al. [35] . Each of the genes are indicated as essential (+), important but not essential (−/+), dispensable (−) for Hcp secretion, or not analyzed (NA) from results of Hcp secretion assays presented in Figure 2A and Figure S1 in File S1. ( B ) RT-PCR analysis. Total RNA extracted from wild-type A. tumefaciens C58 and Δ pro strains grown in AB-MES (pH 5.5) for 6 h at 25°C was treated with RNase-free DNase I followed by RT prior to PCR with gene-specific primers. Genomic DNA and total RNA without RT were positive and negative controls, respectively. The 16S rRNA and the genes atu4329 adjacent to imp operon and atu4353 adjacent to hcp operon were internal controls. PCR products were resolved by 2% agarose gel and stained with ethidium bromide with the analyzed genes as indicated. All analyzed genes with atu number are shown in abbreviation with their last two numbers (e.g. atu4329 is shown as 29 ). ( C ) Western blot analysis. Total proteins isolated from wild-type C58, Δ pro , and Δ imp strains grown in the same growth conditions used for RT-PCR were resolved by 10% or 12% Glycine-SDS-PAGE and analyzed by western blot analysis with specific antibodies as indicated. ActC and RpoA were loading controls. The proteins analyzed and the molecular weight markers are indicated on the left and right, respectively, and with arrows when necessary.
Figure Legend Snippet: The T6SS gene cluster consists of the imp and hcp operons transcribed from divergent promoters. ( A ) The imp operon consisting of 14 genes ( atu4343 to atu4330 ) is in filled colors and the hcp operon encoding nine genes ( atu4344 to atu4352 ) and atu3642 ( vgrG-2 ) encoded elsewhere is in open colors. The genes (indicated with locus names) with homology to conserved T6SS components are designated as tss (type VI secretion) or tag (type VI secretion-associated gene) based on nomenclature proposed by Shalom et al. [35] . Each of the genes are indicated as essential (+), important but not essential (−/+), dispensable (−) for Hcp secretion, or not analyzed (NA) from results of Hcp secretion assays presented in Figure 2A and Figure S1 in File S1. ( B ) RT-PCR analysis. Total RNA extracted from wild-type A. tumefaciens C58 and Δ pro strains grown in AB-MES (pH 5.5) for 6 h at 25°C was treated with RNase-free DNase I followed by RT prior to PCR with gene-specific primers. Genomic DNA and total RNA without RT were positive and negative controls, respectively. The 16S rRNA and the genes atu4329 adjacent to imp operon and atu4353 adjacent to hcp operon were internal controls. PCR products were resolved by 2% agarose gel and stained with ethidium bromide with the analyzed genes as indicated. All analyzed genes with atu number are shown in abbreviation with their last two numbers (e.g. atu4329 is shown as 29 ). ( C ) Western blot analysis. Total proteins isolated from wild-type C58, Δ pro , and Δ imp strains grown in the same growth conditions used for RT-PCR were resolved by 10% or 12% Glycine-SDS-PAGE and analyzed by western blot analysis with specific antibodies as indicated. ActC and RpoA were loading controls. The proteins analyzed and the molecular weight markers are indicated on the left and right, respectively, and with arrows when necessary.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Western Blot, Isolation, SDS Page, Molecular Weight

4) Product Images from "Subtype-associated differences in HIV-1 reverse transcription affect the viral replication"

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Journal: Retrovirology

doi: 10.1186/1742-4690-7-85

Recombinant viruses containing the Gag and Pol domains from HIV-1 subtypes B and C do not have differences in RNA incorporation and GagPol processing . A - Quantitation of viral genomic RNA in virus particles. Virus particles were purified from the culture media of 293T/17 cells transfected with molecular clones of viruses at 48 h post-transfection, treated with DNase I RNase free for 2 h and concentrated by centrifugation through 30% sucrose. RNA was isolated from p24 CA -normalized virus particles, subjected to the reverse transcription with oligo-dT primer and then to quantitative real-time PCR with the primer set specific for positive-strand HIV-1 DNA. The data of analysis of three independent viral preparations were quantified. Each point represents mean RNA copy number ± SD per 1 ng of p24 CA in virus sample. B - Processing of Pr160 GagPol polyprotein-precursor in the virus particles. The virus particles harvested from culture media of transfected 293T/17 cells and purified as in A were analyzed by Western blotting using the antibodies indicated in Materials and Methods. C - Quantification of Western blotting results. Western blotting data from two independent experiments were quantified using ImageJ software. Results show mean grey values of the bands ± SE and are presented as percentage of p24 CA in each virus sample.
Figure Legend Snippet: Recombinant viruses containing the Gag and Pol domains from HIV-1 subtypes B and C do not have differences in RNA incorporation and GagPol processing . A - Quantitation of viral genomic RNA in virus particles. Virus particles were purified from the culture media of 293T/17 cells transfected with molecular clones of viruses at 48 h post-transfection, treated with DNase I RNase free for 2 h and concentrated by centrifugation through 30% sucrose. RNA was isolated from p24 CA -normalized virus particles, subjected to the reverse transcription with oligo-dT primer and then to quantitative real-time PCR with the primer set specific for positive-strand HIV-1 DNA. The data of analysis of three independent viral preparations were quantified. Each point represents mean RNA copy number ± SD per 1 ng of p24 CA in virus sample. B - Processing of Pr160 GagPol polyprotein-precursor in the virus particles. The virus particles harvested from culture media of transfected 293T/17 cells and purified as in A were analyzed by Western blotting using the antibodies indicated in Materials and Methods. C - Quantification of Western blotting results. Western blotting data from two independent experiments were quantified using ImageJ software. Results show mean grey values of the bands ± SE and are presented as percentage of p24 CA in each virus sample.

Techniques Used: Recombinant, Quantitation Assay, Purification, Transfection, Clone Assay, Centrifugation, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Software

5) Product Images from "Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes"

Article Title: Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes

Journal: Nature protocols

doi: 10.1038/nprot.2013.133

The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton X100, 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.
Figure Legend Snippet: The impact of crosslinking on the purification of TF – RNCs. (a,b) Polysome profiles in the absence of crosslinker or after DSP or EDC ex vivo crosslinking and sucrose gradient centrifugation. Depicted are data from experiments in which E. coli MC4100 cells were grown in LB medium and harvested as described in step 1 , option C. The lysate was either crosslinked ex vivo with DSP or EDC (step 7 , option B) or left untreated (step 7 , option A). Undigested (a) and digested (b) lysates were run on a sucrose gradient (step 18 , option B). The digestion was performed with a reduced MNase concentration of 15 U/A 260 to partially retain di- and trisomes for comparison. ‘30S’ and ‘50S’ depict the peaks of the small and large ribosomal subunits, respectively. The monosome peak is labeled with ‘70S’. To compare polysome profiles quantitatively, the curves were normalized to the same area underneath all ribosomal peaks. (c) Gel analysis of the DSP crosslinker titration. 200 ml of E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown and harvested according to step 1 , option A. Cells were resuspended in 2 ml of buffer A (50 mM HEPES pH 7.5, 1 M potassium acetate, 10 mM MgAc 2 , 1 mM PMSF, 1 mM chloramphenicol, 0.4% Triton X100, 0.1% NP-40, 1 mg/ml lysozyme, 2.5 µg/ml RNase-free DNase I). Lysis and purification of TF – RNCs was performed as described including DSP ex vivo crosslinking (step 7 , option B) and sucrose cushion centrifugation (step 18 , option A) with the following exceptions: For crosslinking either 3 mg of DSP (‘1×’), 15 mg of DSP (‘5×’) or DSMO only (‘−‘) were used. Ultracentrifugation was done with 1 M potassium acetate instead of 1 M NaCl in the sucrose cushion buffer causing the high amount of non-crosslinked TF that copelleted without DSP addition (‘−‘). For AP only half of Strep-Tactin slurry and TEV protease were used. Either non-reducing (‘non-red.’) or reducing (‘red.’) sample buffer was used for SDS-PAGE. Gels were stained with coomassie or used for western blotting employing a polyclonal α-TF antibody. Crosslinks are abbreviated with ‘X’. (d) Gel analysis of the EDC crosslinker titration. E. coli MC4100 ∆ tig::Kan + pTrc-tig-TEV-Avi cells were grown in 1 l LB, harvested as described in step 1 , option A, and resuspended in 6 ml of lysis buffer. Ex vivo crosslinking (step 7 , option B) was performed with 2.5 mM (‘0.125×’), 10 mM (‘0.5×’), 20 mM (‘1×’) and 80 mM (‘4×’) EDC. TF–RNCs were purified as described, including sucrose cushion centrifugation (step 18 , option A), eluted from the affinity matrix by boiling in reducing sample buffer, and analyzed by SDS-PAGE or western blotting using polyclonal antibodies against TF and L23. Crosslinks are abbreviated with ‘X’.

Techniques Used: Purification, Ex Vivo, Gradient Centrifugation, Concentration Assay, Labeling, Titration, Lysis, Centrifugation, SDS Page, Staining, Western Blot

6) Product Images from "A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *"

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/ern126

Conservation of the catalytic domain in the AtRNase E/G-like protein. Percentage identities of RNase H, S1, 5′ sensor, and DNase I domains in pair-wise alignments with the A . thaliana protein are shown. Accession nos: P21513 ( E. coli ), BAD03665 ( Oryza sativa ), Q8KAA6 ( Chlorobium tepidum ), P72656 ( Synechocystis sp. PCC 6803). The Zn-link motif co-ordinates a zinc ion involved in quaternary structure ( Callaghan et al. , 2005 a ), S1 domains of A . thaliana and Synechocystis sp. were compared without the plant-specific insert shown in white.
Figure Legend Snippet: Conservation of the catalytic domain in the AtRNase E/G-like protein. Percentage identities of RNase H, S1, 5′ sensor, and DNase I domains in pair-wise alignments with the A . thaliana protein are shown. Accession nos: P21513 ( E. coli ), BAD03665 ( Oryza sativa ), Q8KAA6 ( Chlorobium tepidum ), P72656 ( Synechocystis sp. PCC 6803). The Zn-link motif co-ordinates a zinc ion involved in quaternary structure ( Callaghan et al. , 2005 a ), S1 domains of A . thaliana and Synechocystis sp. were compared without the plant-specific insert shown in white.

Techniques Used: Periodic Counter-current Chromatography

7) Product Images from "Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors"

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064740

LytRS is required for GBAP induction of lrgAB genes. The semi-quantitative RT-PCR shows expression of lrgAB genes in the VI13 (Δ fsrB mutant) and KS19 (Δ fsrB Δ lytRS mutant), in the presence of GBAP. Expression of gelE and gdh were used as positive and negative controls, respectively, of Fsr induction by GBAP and of RNA concentration, respectively. The RNA used for this analysis was previously treated with RNase-free DNase I to remove contaminating DNA.
Figure Legend Snippet: LytRS is required for GBAP induction of lrgAB genes. The semi-quantitative RT-PCR shows expression of lrgAB genes in the VI13 (Δ fsrB mutant) and KS19 (Δ fsrB Δ lytRS mutant), in the presence of GBAP. Expression of gelE and gdh were used as positive and negative controls, respectively, of Fsr induction by GBAP and of RNA concentration, respectively. The RNA used for this analysis was previously treated with RNase-free DNase I to remove contaminating DNA.

Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis, Concentration Assay

8) Product Images from "Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA"

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA

Journal: Biophysical Journal

doi: 10.1529/biophysj.104.052811

( A ) Gel electrophoresis (nondenaturing agarose, 0.75%) of 901 bp dsRNA molecules used in the AFM experiments. A reference dsDNA ladder is shown for comparison in lane 1. After the transcription reaction, we digested the products by both DNase I and an RNase mixture ( lane 2 ). Digestion of the product by the RNase mixture ensures that single-stranded overhangs of the transcription product are removed. Hence, the single remaining band is dsRNA. ( B ) A 1 μ m × 1 μ m AFM image (256 × 256 pixels) of 901 bp dsRNA molecules deposited on mica using polylysine. ( C ) Distribution of lengths measured in the AFM for 901 bp dsRNA molecules. We measured a mean contour length of 258 ± 26 nm, corresponding to a rise per basepair of 0.29 ± 0.03 nm.
Figure Legend Snippet: ( A ) Gel electrophoresis (nondenaturing agarose, 0.75%) of 901 bp dsRNA molecules used in the AFM experiments. A reference dsDNA ladder is shown for comparison in lane 1. After the transcription reaction, we digested the products by both DNase I and an RNase mixture ( lane 2 ). Digestion of the product by the RNase mixture ensures that single-stranded overhangs of the transcription product are removed. Hence, the single remaining band is dsRNA. ( B ) A 1 μ m × 1 μ m AFM image (256 × 256 pixels) of 901 bp dsRNA molecules deposited on mica using polylysine. ( C ) Distribution of lengths measured in the AFM for 901 bp dsRNA molecules. We measured a mean contour length of 258 ± 26 nm, corresponding to a rise per basepair of 0.29 ± 0.03 nm.

Techniques Used: Nucleic Acid Electrophoresis

Related Articles

Clone Assay:

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: An rbcL probe was PCR amplified using primer set 41 + 42 on cloned Brassica napus plastid DNA. .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK).

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: To generate HIV-1 viruses for analysis of reverse transcription, nuclear import, and integration, 293T/17 cells were transfected with different HIV-1 proviral clones alone or with the pcDNA-Env(MLV) plasmid (kindly provided by Nathaniel Landau) at a 4:1 ratio using Metafectene as described earlier (23). .. The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN).

Centrifugation:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: The extracted RNA was added with 2-fold volume of ice-cold 95% ethanol and stored at −70°C for 2 hr before centrifugation at 12,000 g and 15 min at 4°C. .. An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water.

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN). .. Virus particles were further concentrated by centrifugation through a 30% sucrose cushion in PBS at 24,000 RPM in a Beckman SW-28 rotor for 2 h at 4°C.

Amplification:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water. .. PCR cycles were optimized to detect the amplified products before saturation (no more than 30 cycles).

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: An rbcL probe was PCR amplified using primer set 41 + 42 on cloned Brassica napus plastid DNA. .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK).

Synthesized:

Article Title: GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors
Article Snippet: To assess the viral entry of the laboratory-adapted HIV Bal infection, we examined the expression of HIV long terminal repeat (LTR) R/U5 DNA (strong-stop DNA), the first product of HIV reverse transcription, which is synthesized immediately after viral entry and uncoating ( , ). .. Briefly, the viral stock was treated with 10 units/μl of RNase-free DNase I (Roche, Indianapolis, IN) for 30 min prior to its addition to macrophage cultures.

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands). .. For use in the AFM, dsRNA molecules were synthesized using the same HiScribe kit (New England Biolabs).

Cytometry:

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: Sorting was performed by the EMBL Flow Cytometry Core Facility. .. RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions.

Article Title: BRAF V600E and Pten deletion in mice produces a histiocytic disorder with features of Langerhans cell histiocytosis
Article Snippet: Paragraph title: Flow cytometry ... Cells were then incubated with 0.2 units/ml Liberase TM (Roche Diagnostics), 40 units per ml Rnase-free Dnase I (Roche Diagnostics, catalog 04 716 728 001 in HBSS with penicillin and streptomycin for 30 minutes at 37°.

Immunostaining:

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
Article Snippet: Immunocytochemistry and fluorescence in situ hybridization Cells grown on coverslips, and immunostaining was performed after fixation with 1% paraformaldehyde (10 min at room temperature) and permeabilization in 0.2% Triton (20 min on ice) by sequential incubation with primary and Texas red (TR) -labeled secondary antibodies (Vector Laboratories, Burlingame, Calif.) for 30 min each (all solutions in PBS). .. For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind.

SYBR Green Assay:

Article Title: Mx2 is an interferon induced inhibitor of HIV-1 infection
Article Snippet: The virus inoculum was pretreated with 20 U/ml RNase-free DNase I (Roche) for 1 hour at 37 °C in the presence of 6 mM MgCl2 . .. The resulting DNA samples were used as template for quantitative real-time PCR (qPCR) using FastStart Universal SYBR Green Master Mix (Roche) and ABI 7500 Fast PCR system.

Microarray:

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors
Article Snippet: At time zero (immediately after GBAP addition) and after 10 min post-GBAP addition, RNA was extracted from cells and used to synthesize cDNA and perform microarray transcriptional analysis. .. Samples were treated with RNase-free DNase I (Roche) to remove contaminating DNA, and the absence of contaminating DNA was confirmed by PCR.

Incubation:

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
Article Snippet: Immunocytochemistry and fluorescence in situ hybridization Cells grown on coverslips, and immunostaining was performed after fixation with 1% paraformaldehyde (10 min at room temperature) and permeabilization in 0.2% Triton (20 min on ice) by sequential incubation with primary and Texas red (TR) -labeled secondary antibodies (Vector Laboratories, Burlingame, Calif.) for 30 min each (all solutions in PBS). .. For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind.

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: .. The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN). .. Virus particles were further concentrated by centrifugation through a 30% sucrose cushion in PBS at 24,000 RPM in a Beckman SW-28 rotor for 2 h at 4°C.

Article Title: BRAF V600E and Pten deletion in mice produces a histiocytic disorder with features of Langerhans cell histiocytosis
Article Snippet: .. Cells were then incubated with 0.2 units/ml Liberase TM (Roche Diagnostics), 40 units per ml Rnase-free Dnase I (Roche Diagnostics, catalog 04 716 728 001 in HBSS with penicillin and streptomycin for 30 minutes at 37°. .. DNase I was omitted when DNA was to be isolated.

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: The transcription reactions were incubated for 3 h at 37° C. To improve the efficiency of strand annealing, the reactions were heated to 65° C for at least 1 h and cooled down to room temperature by decreasing the temperature by 1.25° C every 5 min in a Mastercycler with heated lid (Eppendorf, Hamburg, Germany). .. The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands).

Immunocytochemistry:

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
Article Snippet: Paragraph title: Immunocytochemistry and fluorescence in situ hybridization ... For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind.

Expressing:

Article Title: SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef
Article Snippet: Analysis of late RT products Virions were produced by co-transfecting 293T cells with NL4-3/Nefstop (1.5 μg) and the pBJ5-based vector expressing SERINC5 (500 ng) or an equimolar amount of empty pBJ5. .. Cell-free virions were treated with RNase-free DNase I (Roche), and used to infect A549/CD4/CXCR4 cells in duplicate in T25 flasks for 14 hrs in the absence or presence of a cocktail of RT inhibitors.

Article Title: GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors
Article Snippet: To assess the viral entry of the laboratory-adapted HIV Bal infection, we examined the expression of HIV long terminal repeat (LTR) R/U5 DNA (strong-stop DNA), the first product of HIV reverse transcription, which is synthesized immediately after viral entry and uncoating ( , ). .. Briefly, the viral stock was treated with 10 units/μl of RNase-free DNase I (Roche, Indianapolis, IN) for 30 min prior to its addition to macrophage cultures.

Modification:

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: The incorporation of labeled nucleotides (biotin-16-UTP and digoxigenin-11-UTP) into 0.4 kb dsRNA was executed by addition of 1–4 molar units of modified UTP to the transcription reactions. .. The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands).

Western Blot:

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN). .. Virus pellets were resuspended either in RPMI medium containing 20 mM HEPES pH 7.4 (for infection) or in PBS for RNA isolation and Western blot analysis.

Derivative Assay:

Article Title: Mx2 is an interferon induced inhibitor of HIV-1 infection
Article Snippet: For analysis of HIV-1 reverse transcription products and 2-LTR circles in infected cells, 1×105 HOS or K562 cells were seeded in 24-well culture plates and infected with a VSV-G pseudotyped HIV-1 reporter virus (CCGW), which is derived from CSGW but encodes a GFP reporter under the control of CMV promoter. .. The virus inoculum was pretreated with 20 U/ml RNase-free DNase I (Roche) for 1 hour at 37 °C in the presence of 6 mM MgCl2 .

Hybridization:

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
Article Snippet: For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind. .. After refixation in 4% paraformaldehyde (10 min at room temperature), samples were equilibrated in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), dehydrated in ethanol (70, 80, and 100% ethanol for 3 min each at −20 °C), air dried, and incubated overnight at 37 °C with the hybridization mixture.

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: Nucleic acid manipulations Procedures for blot analyses, hybridization probe preparation, phosphorimager analyses of band intensities using Aida software and PCR analyses have been described ( ; ). .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK).

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors
Article Snippet: Samples were treated with RNase-free DNase I (Roche) to remove contaminating DNA, and the absence of contaminating DNA was confirmed by PCR. .. RNA integrity was verified using agarose gel electrophoresis of glyoxylated samples (Ambion). cDNA was prepared from RNA using Superscript II Reverse Transcriptase (Invitrogen) with random (N6 ) priming. cDNA was fragmented with dilute DNase I (Roche) and fragments were biotinylated with the BioArray Terminal Labeling Kit (Enzo Life Sciences) prior to hybridization.

Flow Cytometry:

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: Sorting was performed by the EMBL Flow Cytometry Core Facility. .. RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions.

Article Title: BRAF V600E and Pten deletion in mice produces a histiocytic disorder with features of Langerhans cell histiocytosis
Article Snippet: Paragraph title: Flow cytometry ... Cells were then incubated with 0.2 units/ml Liberase TM (Roche Diagnostics), 40 units per ml Rnase-free Dnase I (Roche Diagnostics, catalog 04 716 728 001 in HBSS with penicillin and streptomycin for 30 minutes at 37°.

Ligation:

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: This created sticky ends on the dsRNA molecules which permitted efficient ligation. .. The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands).

Infection:

Article Title: GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors
Article Snippet: To assess the viral entry of the laboratory-adapted HIV Bal infection, we examined the expression of HIV long terminal repeat (LTR) R/U5 DNA (strong-stop DNA), the first product of HIV reverse transcription, which is synthesized immediately after viral entry and uncoating ( , ). .. Briefly, the viral stock was treated with 10 units/μl of RNase-free DNase I (Roche, Indianapolis, IN) for 30 min prior to its addition to macrophage cultures.

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: The 50% tissue culture infective dose of each virus stock was determined by single infection cycle assay using 105 HeLa-CD4-LTR/β-gal (MAGI) indicator cells [ ] for the NL4-3 backbone viruses, or TZM-bl cells for the 1084i backbone viruses, with fourfold serial dilutions of viruses as described previously [ ]. .. The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN).

Article Title: Mx2 is an interferon induced inhibitor of HIV-1 infection
Article Snippet: Paragraph title: Measurement of HIV-1 DNA species in infected cells ... The virus inoculum was pretreated with 20 U/ml RNase-free DNase I (Roche) for 1 hour at 37 °C in the presence of 6 mM MgCl2 .

Reverse Transcription Polymerase Chain Reaction:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: Paragraph title: Total RNA Extraction and RT-PCR ... An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water.

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK). .. The 5′ RACE system (Invitrogen, Paisley, UK) was used on polyA RNA from WT leaves using primer 8 for reverse transcription and following C-tailing PCR amplification with primers 9 and 11, followed by 10 and 12.

Article Title: Aflatoxin Biosynthesis Is a Novel Source of Reactive Oxygen Species—A Potential Redox Signal to Initiate Resistance to Oxidative Stress?
Article Snippet: .. RT-PCR was performed on total RNA treated with RNAse-free DNAse I (Roche Diagnostics, Indianapolis, IN, USA) with primers specific for the coding region of each gene. ..

Generated:

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: The transcription reactions included two PCR products designed so that the two ssRNA molecules generated were complementary except for a few nucleotides at their 5′ ends ( ). .. The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands).

Polymerase Chain Reaction:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water. .. The resulting cDNA was used as the template for PCR reaction.

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: An rbcL probe was PCR amplified using primer set 41 + 42 on cloned Brassica napus plastid DNA. .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK).

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors
Article Snippet: .. Samples were treated with RNase-free DNase I (Roche) to remove contaminating DNA, and the absence of contaminating DNA was confirmed by PCR. .. RNA integrity was verified using agarose gel electrophoresis of glyoxylated samples (Ambion). cDNA was prepared from RNA using Superscript II Reverse Transcriptase (Invitrogen) with random (N6 ) priming. cDNA was fragmented with dilute DNase I (Roche) and fragments were biotinylated with the BioArray Terminal Labeling Kit (Enzo Life Sciences) prior to hybridization.

Article Title: Preclinical Evaluation of In Vitro and In Vivo Antiviral Activities of KCT-01, a New Herbal Formula against Hepatitis B Virus
Article Snippet: They were treated with ribonuclease-free deoxyribonuclease I (Roche, Mannheim, Germany) for 40 min at 37°C to eliminate residual DNA followed by phenol/chloroform extraction, ethanol precipitation, and resuspension with diethyl pyrocarbonate–treated water. .. Extracted RNAs from liver tissues were then subjected to reverse transcription and subsequent PCR reaction.

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: .. The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands). .. The 4.2 kb and 8.3 kb dsRNA molecules were subsequently ligated to the labeled 0.4 kb dsRNA molecules using high concentration T4 DNA ligase ( ).

Article Title: Mx2 is an interferon induced inhibitor of HIV-1 infection
Article Snippet: The virus inoculum was pretreated with 20 U/ml RNase-free DNase I (Roche) for 1 hour at 37 °C in the presence of 6 mM MgCl2 . .. The resulting DNA samples were used as template for quantitative real-time PCR (qPCR) using FastStart Universal SYBR Green Master Mix (Roche) and ABI 7500 Fast PCR system.

Binding Assay:

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: Sorted cells were aliquoted in low binding RNase free tubes and centrifuged at 800 g 10 min at 4°C. .. RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions.

Fluorescence:

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
Article Snippet: Paragraph title: Immunocytochemistry and fluorescence in situ hybridization ... For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind.

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: Paragraph title: Sample collection, fluorescence activated cell sorting and RNA isolation ... RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions.

Mutagenesis:

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions. .. Homozygous lncRNA mutant embryos were manually isolated on ice in PBT under 20x magnification, based on their absence of GFP.

Isolation:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: .. An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water. .. An amount of 1 mg DNA-free RNA was used to synthesize cDNA with SuperScrip III RNase H2 Reverse Transcriptase (Invitrogen, Carlsbad, CA) with specific 3′-primers (Table S2 in File S1).

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: .. RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions. .. Samples were then resuspended in RNase-free H2 O (Ambion) and an aliquot saved for integrity analysis (see later).

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors
Article Snippet: Briefly, RNA was stabilized with RNA protect (Qiagen) and RNA was isolated with RNeasy columns per the manufacturer's instructions (Qiagen). .. Samples were treated with RNase-free DNase I (Roche) to remove contaminating DNA, and the absence of contaminating DNA was confirmed by PCR.

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN). .. Virus pellets were resuspended either in RPMI medium containing 20 mM HEPES pH 7.4 (for infection) or in PBS for RNA isolation and Western blot analysis.

Article Title: BRAF V600E and Pten deletion in mice produces a histiocytic disorder with features of Langerhans cell histiocytosis
Article Snippet: Cells were then incubated with 0.2 units/ml Liberase TM (Roche Diagnostics), 40 units per ml Rnase-free Dnase I (Roche Diagnostics, catalog 04 716 728 001 in HBSS with penicillin and streptomycin for 30 minutes at 37°. .. DNase I was omitted when DNA was to be isolated.

Article Title: Preclinical Evaluation of In Vitro and In Vivo Antiviral Activities of KCT-01, a New Herbal Formula against Hepatitis B Virus
Article Snippet: Paragraph title: 2.11. Isolation and Analysis of Mouse Liver Tissue mRNAs ... They were treated with ribonuclease-free deoxyribonuclease I (Roche, Mannheim, Germany) for 40 min at 37°C to eliminate residual DNA followed by phenol/chloroform extraction, ethanol precipitation, and resuspension with diethyl pyrocarbonate–treated water.

Transfection:

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: To generate HIV-1 viruses for analysis of reverse transcription, nuclear import, and integration, 293T/17 cells were transfected with different HIV-1 proviral clones alone or with the pcDNA-Env(MLV) plasmid (kindly provided by Nathaniel Landau) at a 4:1 ratio using Metafectene as described earlier (23). .. The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN).

Labeling:

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
Article Snippet: Immunocytochemistry and fluorescence in situ hybridization Cells grown on coverslips, and immunostaining was performed after fixation with 1% paraformaldehyde (10 min at room temperature) and permeabilization in 0.2% Triton (20 min on ice) by sequential incubation with primary and Texas red (TR) -labeled secondary antibodies (Vector Laboratories, Burlingame, Calif.) for 30 min each (all solutions in PBS). .. For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind.

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors
Article Snippet: Samples were treated with RNase-free DNase I (Roche) to remove contaminating DNA, and the absence of contaminating DNA was confirmed by PCR. .. RNA integrity was verified using agarose gel electrophoresis of glyoxylated samples (Ambion). cDNA was prepared from RNA using Superscript II Reverse Transcriptase (Invitrogen) with random (N6 ) priming. cDNA was fragmented with dilute DNase I (Roche) and fragments were biotinylated with the BioArray Terminal Labeling Kit (Enzo Life Sciences) prior to hybridization.

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: The incorporation of labeled nucleotides (biotin-16-UTP and digoxigenin-11-UTP) into 0.4 kb dsRNA was executed by addition of 1–4 molar units of modified UTP to the transcription reactions. .. The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands).

Purification:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: .. An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water. .. An amount of 1 mg DNA-free RNA was used to synthesize cDNA with SuperScrip III RNase H2 Reverse Transcriptase (Invitrogen, Carlsbad, CA) with specific 3′-primers (Table S2 in File S1).

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: .. RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions. .. Samples were then resuspended in RNase-free H2 O (Ambion) and an aliquot saved for integrity analysis (see later).

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: Nested PCR primer sets 33 + 34 and 35 + 36 on purified A . thaliana chloroplast DNA, and 37 + 38 and 39 + 40 on total A . thaliana DNA were used for psbA and mt 18S RNA probes, respectively. .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK).

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: .. The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands). .. The 4.2 kb and 8.3 kb dsRNA molecules were subsequently ligated to the labeled 0.4 kb dsRNA molecules using high concentration T4 DNA ligase ( ).

Sequencing:

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK). .. Purified PCR products and plasmids were sequenced with appropriate primers by cycle sequencing using the Big Dye terminator sequencing ready reaction kit (Applied Biosystems, Warrington, UK) and running products on an Applied Biosystems 3100 capillary DNA sequencer.

Construct:

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: Paragraph title: dsRNA constructs ... The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands).

FACS:

Article Title: Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis
Article Snippet: Paragraph title: Sample collection, fluorescence activated cell sorting and RNA isolation ... RNA isolation was performed according to manufacturer’s instructions, including an overnight precipitation step with 1 μL of 10mg/ml glycogen at −800 C. RNA was treated with RNase-free DNase I (Roche) in a 50 μl-volume for 30 min and purified a second time with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer’s instructions.

Nested PCR:

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: Nested PCR primer sets 33 + 34 and 35 + 36 on purified A . thaliana chloroplast DNA, and 37 + 38 and 39 + 40 on total A . thaliana DNA were used for psbA and mt 18S RNA probes, respectively. .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK).

In Situ Hybridization:

Article Title: Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus
Article Snippet: Paragraph title: Immunocytochemistry and fluorescence in situ hybridization ... For simultaneous detection of ZIKV RNA, cells were first immunostained for cellular or viral proteins and then treated for 1 h at 37 °C with RNase-free DNase I (Roche, Indianapolis, Ind.

Plasmid Preparation:

Article Title: SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef
Article Snippet: Analysis of late RT products Virions were produced by co-transfecting 293T cells with NL4-3/Nefstop (1.5 μg) and the pBJ5-based vector expressing SERINC5 (500 ng) or an equimolar amount of empty pBJ5. .. Cell-free virions were treated with RNase-free DNase I (Roche), and used to infect A549/CD4/CXCR4 cells in duplicate in T25 flasks for 14 hrs in the absence or presence of a cocktail of RT inhibitors.

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: To generate HIV-1 viruses for analysis of reverse transcription, nuclear import, and integration, 293T/17 cells were transfected with different HIV-1 proviral clones alone or with the pcDNA-Env(MLV) plasmid (kindly provided by Nathaniel Landau) at a 4:1 ratio using Metafectene as described earlier (23). .. The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN).

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands). .. Using 28iMaI plasmid as a substrate and a T7 minimal primer (both included in the HiScribe kit), a PCR was run to create a 935-bp product with promoter sites for T7 RNA polymerase flanking both ends.

Software:

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: Nucleic acid manipulations Procedures for blot analyses, hybridization probe preparation, phosphorimager analyses of band intensities using Aida software and PCR analyses have been described ( ; ). .. RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK).

Real-time Polymerase Chain Reaction:

Article Title: SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef
Article Snippet: Cell-free virions were treated with RNase-free DNase I (Roche), and used to infect A549/CD4/CXCR4 cells in duplicate in T25 flasks for 14 hrs in the absence or presence of a cocktail of RT inhibitors. .. Genomic DNA was extracted with DNAzol (Life Technologies), and 100 ng of each template DNA was used for quantitative PCR using a LightCycler 96 real-time qPCR system (Roche) and a Kapa SYBR FAST Universal qPCR kit (Kapa Biosystems) according to the manufacturer’s instructions.

Article Title: GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors
Article Snippet: Briefly, the viral stock was treated with 10 units/μl of RNase-free DNase I (Roche, Indianapolis, IN) for 30 min prior to its addition to macrophage cultures. .. Strong-stop DNA was quantified by real-time PCR.

Article Title: Mx2 is an interferon induced inhibitor of HIV-1 infection
Article Snippet: The virus inoculum was pretreated with 20 U/ml RNase-free DNase I (Roche) for 1 hour at 37 °C in the presence of 6 mM MgCl2 . .. The resulting DNA samples were used as template for quantitative real-time PCR (qPCR) using FastStart Universal SYBR Green Master Mix (Roche) and ABI 7500 Fast PCR system.

RNA Extraction:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: Paragraph title: Total RNA Extraction and RT-PCR ... An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water.

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors
Article Snippet: Paragraph title: RNA Extraction and cDNA Synthesis for Microarrays ... Samples were treated with RNase-free DNase I (Roche) to remove contaminating DNA, and the absence of contaminating DNA was confirmed by PCR.

Recombinant:

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: HIV-1 backbone and recombinant virus stocks were prepared by transfecting 293T/17 cells with provirus-encoding plasmids using Metafectene (Biontex, Planegg, Germany). .. The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN).

Agarose Gel Electrophoresis:

Article Title: Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors
Article Snippet: Samples were treated with RNase-free DNase I (Roche) to remove contaminating DNA, and the absence of contaminating DNA was confirmed by PCR. .. RNA integrity was verified using agarose gel electrophoresis of glyoxylated samples (Ambion). cDNA was prepared from RNA using Superscript II Reverse Transcriptase (Invitrogen) with random (N6 ) priming. cDNA was fragmented with dilute DNase I (Roche) and fragments were biotinylated with the BioArray Terminal Labeling Kit (Enzo Life Sciences) prior to hybridization.

Enzyme-linked Immunosorbent Assay:

Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication
Article Snippet: The resulting viruses were then incubated for 2 h at 37°C in a buffer containing 10 mM MgCl2 and 50 U/ml of RNase-free DNase I (Roche, Indianapolis, IN). .. For infection viral titers were normalized to 0.01 or 0.1 pg of p24CA per cell, using a p24 ELISA kit (PerkinElmer, Waltham, MA).

Ethanol Precipitation:

Article Title: Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation
Article Snippet: .. An amount of 5 mg isolated RNA was treated with 10 units of RNase-free DNase I (Roche, Basel, Switzerland) at room temperature for 2 h. The treated RNA was purified by phenol/chloroform extraction, then ethanol precipitation and resuspended in 25 µl DEPC-treated water. .. An amount of 1 mg DNA-free RNA was used to synthesize cDNA with SuperScrip III RNase H2 Reverse Transcriptase (Invitrogen, Carlsbad, CA) with specific 3′-primers (Table S2 in File S1).

Article Title: Preclinical Evaluation of In Vitro and In Vivo Antiviral Activities of KCT-01, a New Herbal Formula against Hepatitis B Virus
Article Snippet: .. They were treated with ribonuclease-free deoxyribonuclease I (Roche, Mannheim, Germany) for 40 min at 37°C to eliminate residual DNA followed by phenol/chloroform extraction, ethanol precipitation, and resuspension with diethyl pyrocarbonate–treated water. .. Extracted RNAs from liver tissues were then subjected to reverse transcription and subsequent PCR reaction.

Produced:

Article Title: SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef
Article Snippet: Analysis of late RT products Virions were produced by co-transfecting 293T cells with NL4-3/Nefstop (1.5 μg) and the pBJ5-based vector expressing SERINC5 (500 ng) or an equimolar amount of empty pBJ5. .. Cell-free virions were treated with RNase-free DNase I (Roche), and used to infect A549/CD4/CXCR4 cells in duplicate in T25 flasks for 14 hrs in the absence or presence of a cocktail of RT inhibitors.

Concentration Assay:

Article Title: Single-Molecule Measurements of the Persistence Length of Double-Stranded RNA
Article Snippet: The dsRNA transcription reactions were further treated with five units of RNase-free DNase I (Roche Diagnostics, Almere, The Netherlands) for 1 h at 37° C to remove the PCR products and purified using a RNeasy MinElute Cleanup Kit (Qiagen Benelux, Venlo, The Netherlands). .. The 4.2 kb and 8.3 kb dsRNA molecules were subsequently ligated to the labeled 0.4 kb dsRNA molecules using high concentration T4 DNA ligase ( ).

Staining:

Article Title: Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes
Article Snippet: Phenylmethylsulfonylfluoride (PMSF, Roth, 6367) Triton X100 (Roth, 3051) Nonidet P 40 Substitute (NP-40, Sigma, 74385) RNase-free DNase I (Roche, 04716728001) HCl 37% (wt/wt) (VWR, 20252.335) CAUTION Hydrochloric acid is corrosive. .. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, Thermo Scientific, 22980) Glycine (Roth, 3790) NaHCO3 (Roth, 6885) Superase•In RNase inhibitor (Ambion, AM2696) Micrococcal nuclease (Nuclease S7) (Roche, 10107921001 or ) Ethylene glycol tetraacetic acid (EGTA, AppliChem, A0878) Sucrose (Sigma, 16104) Strep-Tactin Sepharose 50% suspension (IBA, 2-1201-025) DNA, sodium salt from salmon testes (Sigma-Aldrich, D1626) Kanamycin (Applichem, A1493) Isopropyl β-D-1-thiogalactopyranoside (IPTG, Roth, CN08) Colloidal coomassie staining solution (Roth, Roti-Blue quick, 4829) Imidazole (Roth, 3899) CAUTION Imidazole is corrosive.

Variant Assay:

Article Title: A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis *
Article Snippet: RNA for RT-PCR experiments was treated with RNase-free DNase I (Roche, Lewes, UK). .. The exon 4 splice variant was detected by RT-PCR with primers 13 and 14.

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  • 99
    Roche dnase i
    <t>DNase</t> I hypersensitivity mapping upstream of XIST . Below the schematic of the XIST region (see legend in Figure 1) are Southern blots of DNA from ES-10, an Xa hybrid, and an Xi hybrid. Small rectangular boxes are probes for Southern blotting of overlapping restriction fragments (from left to right: Pst I (13.1 kb); Bsm I (10.3 kb); Nde I (7.2 kb); Sap I (6.7 kb); Spe I (6.3 kb); Sca I (3.8 kb)). All experiments were carried out at least twice. One HS site, highlighted by the open arrow, was detected just upstream of the transcriptional start site, within the Spe I fragment, at somewhat variable positions in the three cell lines. Another HS site was found within the Bsm I fragment approximately 65 kb upstream of the XIST transcription start in both Xa and Xi hybrids (grey arrow). No other DNase I hypersensitive sites were observed.
    Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Roche rnase free certified dnase i
    Melting curve (A) amplified at various annealing temparature and gel-electrophoresis pattern (B) of 18SrRNA amplified at 60° of annealing temperature by real-time PCR using the RT product of the cell lysate treated with 0.16 U/μl <t>RNase-free</t> <t>DNase</t> I for 30 min. Lane 1: 100-bp Ladder size marker; Lane 2: amplification product of 18SrRNA (120 bp) in gel-electrophoresis pattern.
    Rnase Free Certified Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche recombinant dnase i
    DNase activity of Mo -CBP 2. The pUC18 plasmid (500.0 ng) of E. coli was incubated with Mo -CBP 2 (500.0 ng); BSA (500.0 ng) and 0.05 M Tris-HCl buffer, pH 7.4 (negative controls); and the recombinant <t>DNase</t> I (2 units, positive control) for 1 h and loaded in 1% (m/mL) agarose gel electrophoresis. Gel was stained with ethidium bromide and observed under UV light.
    Recombinant Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNase I hypersensitivity mapping upstream of XIST . Below the schematic of the XIST region (see legend in Figure 1) are Southern blots of DNA from ES-10, an Xa hybrid, and an Xi hybrid. Small rectangular boxes are probes for Southern blotting of overlapping restriction fragments (from left to right: Pst I (13.1 kb); Bsm I (10.3 kb); Nde I (7.2 kb); Sap I (6.7 kb); Spe I (6.3 kb); Sca I (3.8 kb)). All experiments were carried out at least twice. One HS site, highlighted by the open arrow, was detected just upstream of the transcriptional start site, within the Spe I fragment, at somewhat variable positions in the three cell lines. Another HS site was found within the Bsm I fragment approximately 65 kb upstream of the XIST transcription start in both Xa and Xi hybrids (grey arrow). No other DNase I hypersensitive sites were observed.

    Journal: BMC Molecular Biology

    Article Title: Identification of regulatory elements flanking human XIST reveals species differences

    doi: 10.1186/1471-2199-11-20

    Figure Lengend Snippet: DNase I hypersensitivity mapping upstream of XIST . Below the schematic of the XIST region (see legend in Figure 1) are Southern blots of DNA from ES-10, an Xa hybrid, and an Xi hybrid. Small rectangular boxes are probes for Southern blotting of overlapping restriction fragments (from left to right: Pst I (13.1 kb); Bsm I (10.3 kb); Nde I (7.2 kb); Sap I (6.7 kb); Spe I (6.3 kb); Sca I (3.8 kb)). All experiments were carried out at least twice. One HS site, highlighted by the open arrow, was detected just upstream of the transcriptional start site, within the Spe I fragment, at somewhat variable positions in the three cell lines. Another HS site was found within the Bsm I fragment approximately 65 kb upstream of the XIST transcription start in both Xa and Xi hybrids (grey arrow). No other DNase I hypersensitive sites were observed.

    Article Snippet: Nuclei were digested with an increasing amount of DNase I (10 U/μl, RNase free, Roche) (i.e. 1/128, 1/64, 1/32, 1/16, 1/8, 1/4 U/μl) at 37°C for 20 minutes.

    Techniques: Southern Blot

    Mapping DNase I hypersensitive sites downstream of XIST . A schematic of the mouse (A) and human (B) Xist / XIST genes and surrounding regulatory elements shows exons (black boxes) for Xist / XIST and other genes in the region (arrows indicate the direction of transcription). Grey dots show the location of conserved blocks between mouse and human [ 15 ], and triangles mark DNase I hypersensitive (DHS) sites (filled triangles - sites in undifferentiated cells; empty triangles - sites in differentiated cells; HS1, HS5 - HS7 [ 40 ], HS2 - HS4 [ 46 , 47 ], HS8 - HS15 [ 55 ]). DNA from nuclei of ES-10, L1.10.1 and Xa and Xi-containing hybrids exposed to DNaseI was digested with restriction enzymes ( Sca I (10 kb); Sap I/ Blp I (10.2 kb); Nsi I (11.1 kb); Xmn I (9.3 kb); Nco I (11.1 kb) and Bam HI (6.4 kb)) for Southern analysis. All experiments were carried out at least twice and a representative Southern blot is shown for each fragment including a lane with no DNase I (parental fragment shown as black arrow) followed by six lanes of increasing amount of DNase I treatment. One HS site (grey arrow), was detected in the Sap I/ Blp I fragment of ES-10 cells, with a size of ~7 kb. No HS site was detected in the Xmn I fragment which encompasses the four previously reported transcription start sites of TSIX [ 32 ]. No HS site was detected in either the Xa or Xi hybrid downstream of XIST .

    Journal: BMC Molecular Biology

    Article Title: Identification of regulatory elements flanking human XIST reveals species differences

    doi: 10.1186/1471-2199-11-20

    Figure Lengend Snippet: Mapping DNase I hypersensitive sites downstream of XIST . A schematic of the mouse (A) and human (B) Xist / XIST genes and surrounding regulatory elements shows exons (black boxes) for Xist / XIST and other genes in the region (arrows indicate the direction of transcription). Grey dots show the location of conserved blocks between mouse and human [ 15 ], and triangles mark DNase I hypersensitive (DHS) sites (filled triangles - sites in undifferentiated cells; empty triangles - sites in differentiated cells; HS1, HS5 - HS7 [ 40 ], HS2 - HS4 [ 46 , 47 ], HS8 - HS15 [ 55 ]). DNA from nuclei of ES-10, L1.10.1 and Xa and Xi-containing hybrids exposed to DNaseI was digested with restriction enzymes ( Sca I (10 kb); Sap I/ Blp I (10.2 kb); Nsi I (11.1 kb); Xmn I (9.3 kb); Nco I (11.1 kb) and Bam HI (6.4 kb)) for Southern analysis. All experiments were carried out at least twice and a representative Southern blot is shown for each fragment including a lane with no DNase I (parental fragment shown as black arrow) followed by six lanes of increasing amount of DNase I treatment. One HS site (grey arrow), was detected in the Sap I/ Blp I fragment of ES-10 cells, with a size of ~7 kb. No HS site was detected in the Xmn I fragment which encompasses the four previously reported transcription start sites of TSIX [ 32 ]. No HS site was detected in either the Xa or Xi hybrid downstream of XIST .

    Article Snippet: Nuclei were digested with an increasing amount of DNase I (10 U/μl, RNase free, Roche) (i.e. 1/128, 1/64, 1/32, 1/16, 1/8, 1/4 U/μl) at 37°C for 20 minutes.

    Techniques: Southern Blot

    Localization of the -65 DNase I hypersensitive site upstream of XIST . A. Using an Xa hybrid to limit background from cross-hybridization to human material, two additional restriction digests were performed on different DNA preparations after increasing DNase I treatments to refine the location of the -65 site. The Bgl II fragment is 9.3 kb and the fragment resulting from HS and restriction digestion is 5 kb (grey arrow, left panel). The Sca I fragment is 11.8 kb and the fragment resulting from HS and restriction digestion is 8 kb (grey arrow, right panel). Dot-plot analyses http://mulan.dcode.org/ showing comparison of sequence upstream of XIST between cow (38 kb) and human (92 kb) (panel B) and mouse (10 kb) and human (92 kb) (panel C).

    Journal: BMC Molecular Biology

    Article Title: Identification of regulatory elements flanking human XIST reveals species differences

    doi: 10.1186/1471-2199-11-20

    Figure Lengend Snippet: Localization of the -65 DNase I hypersensitive site upstream of XIST . A. Using an Xa hybrid to limit background from cross-hybridization to human material, two additional restriction digests were performed on different DNA preparations after increasing DNase I treatments to refine the location of the -65 site. The Bgl II fragment is 9.3 kb and the fragment resulting from HS and restriction digestion is 5 kb (grey arrow, left panel). The Sca I fragment is 11.8 kb and the fragment resulting from HS and restriction digestion is 8 kb (grey arrow, right panel). Dot-plot analyses http://mulan.dcode.org/ showing comparison of sequence upstream of XIST between cow (38 kb) and human (92 kb) (panel B) and mouse (10 kb) and human (92 kb) (panel C).

    Article Snippet: Nuclei were digested with an increasing amount of DNase I (10 U/μl, RNase free, Roche) (i.e. 1/128, 1/64, 1/32, 1/16, 1/8, 1/4 U/μl) at 37°C for 20 minutes.

    Techniques: Hybridization, Sequencing

    Melting curve (A) amplified at various annealing temparature and gel-electrophoresis pattern (B) of 18SrRNA amplified at 60° of annealing temperature by real-time PCR using the RT product of the cell lysate treated with 0.16 U/μl RNase-free DNase I for 30 min. Lane 1: 100-bp Ladder size marker; Lane 2: amplification product of 18SrRNA (120 bp) in gel-electrophoresis pattern.

    Journal: Reproductive biology and endocrinology : RB & E

    Article Title: Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation

    doi: 10.1186/1477-7827-3-59

    Figure Lengend Snippet: Melting curve (A) amplified at various annealing temparature and gel-electrophoresis pattern (B) of 18SrRNA amplified at 60° of annealing temperature by real-time PCR using the RT product of the cell lysate treated with 0.16 U/μl RNase-free DNase I for 30 min. Lane 1: 100-bp Ladder size marker; Lane 2: amplification product of 18SrRNA (120 bp) in gel-electrophoresis pattern.

    Article Snippet: DNase digestion and reverse transcript Cell lysates were supplemented with the different concentrations (final concentrations of 0.04 U/μl and 0.08 U/μl) of DNase I provided in the cells-to-cDNA II kit or with two concentrations (final concentrations of 0.08 U/μl and 0.16 U/μl) of RNase-free certified DNase I with 10 U/μl (Roche, Penzberg, Germany).

    Techniques: Amplification, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Marker

    Amplification curves (A) and melting curves (B) of 18SrRNA amplified by real-time PCR using the RT-minus product of the cell lysate treated for 15, 30 and 60 min with 0.08 U/μl RNase-free DNase I.

    Journal: Reproductive biology and endocrinology : RB & E

    Article Title: Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation

    doi: 10.1186/1477-7827-3-59

    Figure Lengend Snippet: Amplification curves (A) and melting curves (B) of 18SrRNA amplified by real-time PCR using the RT-minus product of the cell lysate treated for 15, 30 and 60 min with 0.08 U/μl RNase-free DNase I.

    Article Snippet: DNase digestion and reverse transcript Cell lysates were supplemented with the different concentrations (final concentrations of 0.04 U/μl and 0.08 U/μl) of DNase I provided in the cells-to-cDNA II kit or with two concentrations (final concentrations of 0.08 U/μl and 0.16 U/μl) of RNase-free certified DNase I with 10 U/μl (Roche, Penzberg, Germany).

    Techniques: Amplification, Real-time Polymerase Chain Reaction

    Amplification curves (A) and melting curves (B) of 18SrRNA amplified by real-time PCR using the RT – minus product of the cell lysate treated for 15, 30 and 60 min with 0.16 U/μl RNase-free DNase I.

    Journal: Reproductive biology and endocrinology : RB & E

    Article Title: Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation

    doi: 10.1186/1477-7827-3-59

    Figure Lengend Snippet: Amplification curves (A) and melting curves (B) of 18SrRNA amplified by real-time PCR using the RT – minus product of the cell lysate treated for 15, 30 and 60 min with 0.16 U/μl RNase-free DNase I.

    Article Snippet: DNase digestion and reverse transcript Cell lysates were supplemented with the different concentrations (final concentrations of 0.04 U/μl and 0.08 U/μl) of DNase I provided in the cells-to-cDNA II kit or with two concentrations (final concentrations of 0.08 U/μl and 0.16 U/μl) of RNase-free certified DNase I with 10 U/μl (Roche, Penzberg, Germany).

    Techniques: Amplification, Real-time Polymerase Chain Reaction

    DNase activity of Mo -CBP 2. The pUC18 plasmid (500.0 ng) of E. coli was incubated with Mo -CBP 2 (500.0 ng); BSA (500.0 ng) and 0.05 M Tris-HCl buffer, pH 7.4 (negative controls); and the recombinant DNase I (2 units, positive control) for 1 h and loaded in 1% (m/mL) agarose gel electrophoresis. Gel was stained with ethidium bromide and observed under UV light.

    Journal: Frontiers in Microbiology

    Article Title: A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production

    doi: 10.3389/fmicb.2017.00980

    Figure Lengend Snippet: DNase activity of Mo -CBP 2. The pUC18 plasmid (500.0 ng) of E. coli was incubated with Mo -CBP 2 (500.0 ng); BSA (500.0 ng) and 0.05 M Tris-HCl buffer, pH 7.4 (negative controls); and the recombinant DNase I (2 units, positive control) for 1 h and loaded in 1% (m/mL) agarose gel electrophoresis. Gel was stained with ethidium bromide and observed under UV light.

    Article Snippet: Recombinant DNase I (2 units, RNase-free, Roche), BSA (500.0 ng), and 0.05 M Tris-HCl buffer, pH 7.4, all equally incubated with the plasmid, were used as controls.

    Techniques: Activity Assay, Plasmid Preparation, Incubation, Recombinant, Positive Control, Agarose Gel Electrophoresis, Staining