dnase  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding

doi: 10.1093/nar/gkr640

Figure Lengend Snippet: ( A ) DNase I footprinting analysis of the binding of wild-type and N-terminally truncated derivatives of SimR to the simR–simX intergenic region. A DNA fragment containing the simR–simX intergenic region, 5′-end labelled on either the upper strand (left panel) or the lower strand (right panel), was exposed to DNase I in the presence of saturating concentrations of SimR protein (200 nM for wild-type SimR, SimRΔN10 and SimRΔN15; 400 nM for SimRΔN22 and SimRΔN25). The sequencing ladders were generated by subjecting the probes to Maxam-Gilbert G+A chemical sequencing. Regions protected from DNase I cleavage (operators O X and O R ) by wild-type SimR are indicated by solid vertical bars, and those protected by the N-terminally truncated SimR derivatives are indicated by open bars. Inverted repeats within the DNase I protected regions are indicated by convergent arrows. ( B ) Sequence of the simR–simX intergenic region summarizing the DNase I footprinting data. Regions protected by wild-type SimR are indicated by solid lines, and those protected by the N-terminally truncated SimR derivatives are indicated by dotted lines. Also indicated are the simRp and simXp transcription start points and putative −10 sequences, the simR and simX ribosome-binding sites (RBS), and the imperfect inverted repeats within the footprints.

Article Snippet: DNase I footprinting Templates for DNase I footprinting were amplified by PCR using one unlabelled primer and one primer 5′-end labelled using [γ32 -P] ATP and T4 polynucleotide kinase (New England Biolabs).

Techniques: Footprinting, Binding Assay, Sequencing, Generated

Journal: PLoS Genetics

Article Title: Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription

doi: 10.1371/journal.pgen.1002585

Figure Lengend Snippet: Non-overlapping regulatory modules within angptl4 intron 3 confer liver, islet, and enterocyte-specific reporter expression. (A) Depiction of the 6 dpf zebrafish showing liver (li, green), intestine (in, blue), swim bladder (sb, grey), and muscle (mu, grey), with the fish oriented anterior (a) to the left and posterior (p) to the right. The opposite orientation reveals the exocrine pancreas (pa, yellow) and islet (is, orange). (B) Scaled schematic of the zebrafish angptl4 locus and non-coding DNA assayed for regulatory potential. Modules are color coded according to the tissues in which they confer expression. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown in parentheses next to truncation labels. (C–N) Representative images of GFP reporter expression in mosaic (column 1) and F 1 stable (column 2) animals driven by each non-coding DNA region (rows). Scale bars = 100 µm; li = liver, is = islet, in = intestine, sb = swim bladder. Colored arrowheads indicate tissue with specific reporter expression. (C–D) Full-length intron 3 (in3; 2,136 bp) is sufficient to promote expression of the reporter in the liver, islet (D, inset, scale bar = 50 µm), and intestine. (E–F) Truncation in3.1 (1,219 bp) confers expression in the liver. (G–H) Truncation in3.2 (701 bp) confers expression in both the intestine and islet (H, inset). Inset scale bar = 50 µm. (I–J) Truncation in3.3 (387 bp) confers islet expression. A transverse section (inset, J) reveals islet expression (nuclei stained with DAPI). Inset scale bar = 50 µm. (K–L) Truncation in3.4 (316 bp) confers intestinal expression. Insets in panels K and L contain transverse sections showing expression localized to the intestinal epithelium (nuclei stained with DAPI). Inset scale bar = 25 µm. The dotted lines in panels D, G, H, and I outline the pancreas. The white arrows in panels H, K, and L mark the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). (M–N) Cells expressing GFP driven by the in3.4 regulatory module colocalize with a marker (4E8, red, white arrow) of the brush border of absorptive enterocytes, but fail to co-localize with marker for secretory cells (2F11, red, asterisk). Nuclei stained with DAPI. Scale bars = 5 µm. (O) Intercross of Tg(in3.2-Mmu.Fos:tdTomato) with β-cell specific reporter line ( Tg(ins:CFP-NTR) s892 ) show colocalization of tdTomato and CFP in the islet. Scale bars = 10 µm. (P) Quantitative PCR shows that the in3.4 module and the angptl4 promoter (TATA box), but not the in3.3 module, are hypersensitive to DNase I cleavage in intestinal epithelial cells isolated from adult zebrafish. Asterisks denote P-value

Article Snippet: Nuclei were incubated with various concentrations of Dnase I (0–1.5 units, NEB) for 10 minutes at 37°C.

Techniques: Expressing, Fluorescence In Situ Hybridization, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation

Journal: bioRxiv

Article Title: A Novel Virus Discovered in the Yeast Pichia membranifaciens

doi: 10.1101/2022.01.05.475065

Figure Lengend Snippet: Extrachromosomal nucleic acids within P. membranifaciens are double stranded RNA molecules. Agarose gel electrophoresis of cellulose column purified nucleic acids from P. membranifaciens NCYC333 with DNase I (lane 1) and ShortCut® RNase III (lane 2).

Article Snippet: DNase I (New England Biolabs) digestion was performed as directed by the manufacturer in 1X reaction buffer for 10 min at 37°C. the reaction was stopped by incubation at 75°C for 10 min. All digested products were analyzed by agarose gel electrophoresis with ethidium bromide staining.

Techniques: Agarose Gel Electrophoresis, Purification

Journal: bioRxiv

Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

doi: 10.1101/2021.03.31.437857

Figure Lengend Snippet: Fluorescence image of fragmented DNA remaining after digestion by DNase I diffusing through microfluidic channels. Distance from reservoir inlet is 1.1mm.

Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

doi: 10.1101/2021.03.31.437857

Figure Lengend Snippet: DNA (at high density) fragmented on a surface by DNase I. Distance from inlet is 8.7mm.

Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

Techniques:

Journal: bioRxiv

Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

doi: 10.1101/2021.03.31.437857

Figure Lengend Snippet: Schematic of stamping method for fragmenting surface-adsorbed. A PDMS stamp in the form of a grating is ‘inked’ with DNase I cuttting enzyme and is brought into contact with a surface on which DNA molecules have been deposited.

Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

Techniques:

Journal: bioRxiv

Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

doi: 10.1101/2021.03.31.437857

Figure Lengend Snippet: Fluorescence image of SyBr Gold labeled DNA. Upper left area was covered with a solution containing 0.095U/μl of DNase I in NEB DNase I Reaction Buffer and shows effective digestion of DNA in that region.

Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

Techniques: Fluorescence, Labeling