m0210  (New England Biolabs)

Journal: Nature

Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

doi: 10.1038/nature25449

Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

Techniques: Inhibition, Immunofluorescence, Staining

Journal: Nucleic Acids Research

Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

doi: 10.1093/nar/gks316

Figure Lengend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

Techniques: Purification, Polymerase Chain Reaction, Construct

Journal: Applied and Environmental Microbiology

Article Title: Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

doi: 10.1128/AEM.67.12.5780-5790.2001

Figure Lengend Snippet: Quantitative analysis of functional gene arrays. (A) Relationship of hybridization signal intensity to DNA target concentration from a single pure culture. Genomic DNA from nirS -containing P. stutzeri E4-2 was labeled with Cy5 and hybridized to the microarrays at the following target concentrations: 0.5, 1, 2.5, 5, 10, 25, 50, and 100 ng. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration. (B) Relationship of hybridization signal intensity to DNA target concentration using a mixture of target DNAs. The PCR products from the following nine strains were mixed together in different quantities (in picograms): E4-2 ( nirS ), 1,000; G179 ( nirK ), 500; wc301–37 ( amoA ), 250; ps-47 ( amoA ), 125; pB49 ( nirS ), 62.5; Y32K ( nirK ), 31.3; wA15 ( nirS ), 15.6; ps-80 ( amoA ), 7.8; wB54 ( nirK ), 3.9. All of these genes are less than 80% identical. The mixed templates were labeled with Cy5. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration for each strain. (C) Effects of probe DNA size and composition on hybridization signal intensity. Microarrays contained DNA fragments (200 ng μl −1 ) of different sizes amplified from different regions in the S. oneidensis MR-1 genome. The target DNA was prepared by labeling MR-1 genomic DNA with Cy5 using the Klenow fragment with random hexamer primers. For panels A and B, the data points are mean values derived from three independent microarray slides, with three replicates on each slide (a total of nine data points). Error bars showing the standard deviations are presented.

Article Snippet: Each 40-μl labeling reaction mixture contained denatured genomic DNA; 1.5 μg of random hexamers (Gibco BRL, Gaithersburg, Md.); 1× EcoPol buffer (New England Biolabs, Beverly, Mass.); 50 μM dATP, dTTP, and dGTP; 20 μM dCTP; 10 μM Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.); 2.5 mM dithiothreitol; and 10 U of the large Klenow fragment of DNA polymerase I (New England Biolabs).

Techniques: Functional Assay, Hybridization, Concentration Assay, Labeling, Transformation Assay, Polymerase Chain Reaction, Amplification, Random Hexamer Labeling, Derivative Assay, Microarray

Journal: Applied and Environmental Microbiology

Article Title: Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

doi: 10.1128/AEM.67.12.5780-5790.2001

Figure Lengend Snippet: The normalized distribution of signal intensity levels for nirS, nirK , and amoA genes as determined by microarray-based analysis of community DNA from marine sediment (W303) and surface soil (O22) environments. Bulk community DNA isolated from two different environmental samples was directly labeled with Cy5 using Klenow fragment with random hexamer primers and hybridized with the microarrays at 65°C in separate experiments. The hybridization signal intensity for each gene is presented. Shaded bars represent probes from environmental clones, and striped boxes represent pmo A probes from environmental clones. Open bars designate probes from pure cultures. The data represent mean values obtained from nine replicates after subtracting background hybridization to yeast genes. For the nirS graphs, bars 1 to 42 correspond to individual genes S1-S42 (see assigned designation in Table 1 on the web site). Similarly, bars 1 to 18 for nirK correspond to genes K1 to K18, and bars 1 to 22 for amoA and pmoA represent genes M1 to M22 (see our table on the website cited in Materials and Methods). The standard deviation of signal intensity is indicated on the top of each bar.

Article Snippet: Each 40-μl labeling reaction mixture contained denatured genomic DNA; 1.5 μg of random hexamers (Gibco BRL, Gaithersburg, Md.); 1× EcoPol buffer (New England Biolabs, Beverly, Mass.); 50 μM dATP, dTTP, and dGTP; 20 μM dCTP; 10 μM Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.); 2.5 mM dithiothreitol; and 10 U of the large Klenow fragment of DNA polymerase I (New England Biolabs).

Techniques: Microarray, Isolation, Environmental Sampling, Labeling, Random Hexamer Labeling, Hybridization, Clone Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology

doi: 10.1093/nar/gkq512

Figure Lengend Snippet: Analysis of the overhang types created by ZFNs. ( A ) Scheme to determine ZFN overhangs. A supercoiled plasmid with a ZFN cleavage site is cut by a titration of in vitro transcribed and translated ZFNs. ZFN-linearized plasmids are purified by gel electrophoresis, 5′ overhangs filled in with Klenow polymerase (grey nucleotides), and the resulting blunt ends ligated. The mixture is subjected to high-throughput DNA sequencing. ( B ) Overhang types generated by a control restriction enzyme (HindIII) and three of the ZFN pairs used in this work. For clarity, only one DNA strand is shown. Enzyme binding sites are shown in grey; only the flanking three nucleotides are shown for ZFN binding sites. Primary cleavage sites, black triangles; secondary and tertiary cleavage sites, dark and light grey triangles, respectively; deletions, Δ. Microhomology within the target site can prevent unambiguous deduction of the overhang type. In such situations the possible overhangs are shown as joined triangles. Either of the two indicated thymidine residues may have been deleted after HindIII digestion.

Article Snippet: Linearized plasmids were gel purified by agarose gel electrophoresis and incubated for 30 min at 37°C with 0.05 U Klenow DNA polymerase (New England Biolabs) in 1 × Buffer 2, plus 50 µM dNTPs.

Techniques: Plasmid Preparation, Titration, In Vitro, Purification, Nucleic Acid Electrophoresis, High Throughput Screening Assay, DNA Sequencing, Generated, Binding Assay