doubly biotinylated λ dna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher doubly biotinylated λ dna
    Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on  λ  DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on  λ  DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647  signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on  λ  DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on  λ  DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on  λ  DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on  λ  DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on  λ  DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on  λ  DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.
    Doubly Biotinylated λ Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication"

    Article Title: Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

    Journal: bioRxiv

    doi: 10.1101/789578

    Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on  λ  DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on  λ  DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647  signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on  λ  DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on  λ  DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on  λ  DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on  λ  DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on  λ  DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on  λ  DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.
    Figure Legend Snippet: Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on λ DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on λ DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647 signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on λ DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on λ DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on λ DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on λ DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on λ DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on λ DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.

    Techniques Used: Staining, Fluorescence, Migration, Imaging, Concentration Assay

    Histone dynamics during DNA licensing in HSS. (A) Schematic of the experimental set-up for real-time single-molecule imaging of nucleosome dynamics during replication in  Xenopus leavis  egg extracts.  λ  DNA containing fluorescent nucleosomes (one of the four histones labelled fluorescently) is stretched under flow and tethered at both ends to the functionalized glass surface of a microfluidic flow cell. The immobilized DNA is licensed in high-speed supernatant (HSS). Bidirectional replication is initiated upon introduction of nucleoplasmic extract (NPE) supplemented with a fluorescent fusion protein Fen1-KikGR, which decorates replication bubbles and allows progression of replication forks to be tracked in real time. Cy5- or Alexa647-labelled histones within immobilized nucleosomal templates are imaged with a 640-nm laser at each stage. Replication fork progression is visualized in NPE using a 488-nm laser. (B and C) Kymograms and corresponding intensity profiles for fluorescent  λ  nucleosomes during incubation in HSS. Nucleosomes labelled at H2A-K119C with Cy5 and H2B-T112C with AlexaFluor647 (B) show faster loss of fluorescence than nucleosomes labelled at H3-K36C with Cy5, H3-T80C with AlexaFluor647 and H4-E63C with AlexaFluor647 (C). (D) Plot showing the mean loss of fluorescent signal for  λ  nucleosomes (H2A-K119 Cy5 , H2B-T112C A647 , H3-K36C Cy5 , H3-T80C A647  and H4-E63C A647 ) during incubation in HSS. Over 100 molecules were analyzed for each histone template. Individual fluorescence decay traces were normalized to background (‘0’) and maximum value of fluorescence (‘1’). A mean fluorescence value and standard deviation were calculated and plotted for each time point. The mean value traces were then fitted to a single exponential function. (E) Summary of the fluorescence decay rate constants ( K ) and half-lives ( t 0.5) extracted from the single exponential fit to the data presented in panel C. See Table S2 for detailed fitting parameters.
    Figure Legend Snippet: Histone dynamics during DNA licensing in HSS. (A) Schematic of the experimental set-up for real-time single-molecule imaging of nucleosome dynamics during replication in Xenopus leavis egg extracts. λ DNA containing fluorescent nucleosomes (one of the four histones labelled fluorescently) is stretched under flow and tethered at both ends to the functionalized glass surface of a microfluidic flow cell. The immobilized DNA is licensed in high-speed supernatant (HSS). Bidirectional replication is initiated upon introduction of nucleoplasmic extract (NPE) supplemented with a fluorescent fusion protein Fen1-KikGR, which decorates replication bubbles and allows progression of replication forks to be tracked in real time. Cy5- or Alexa647-labelled histones within immobilized nucleosomal templates are imaged with a 640-nm laser at each stage. Replication fork progression is visualized in NPE using a 488-nm laser. (B and C) Kymograms and corresponding intensity profiles for fluorescent λ nucleosomes during incubation in HSS. Nucleosomes labelled at H2A-K119C with Cy5 and H2B-T112C with AlexaFluor647 (B) show faster loss of fluorescence than nucleosomes labelled at H3-K36C with Cy5, H3-T80C with AlexaFluor647 and H4-E63C with AlexaFluor647 (C). (D) Plot showing the mean loss of fluorescent signal for λ nucleosomes (H2A-K119 Cy5 , H2B-T112C A647 , H3-K36C Cy5 , H3-T80C A647 and H4-E63C A647 ) during incubation in HSS. Over 100 molecules were analyzed for each histone template. Individual fluorescence decay traces were normalized to background (‘0’) and maximum value of fluorescence (‘1’). A mean fluorescence value and standard deviation were calculated and plotted for each time point. The mean value traces were then fitted to a single exponential function. (E) Summary of the fluorescence decay rate constants ( K ) and half-lives ( t 0.5) extracted from the single exponential fit to the data presented in panel C. See Table S2 for detailed fitting parameters.

    Techniques Used: Imaging, Incubation, Fluorescence, Standard Deviation

    Assembly of fluorescent nucleosomes on λ DNA. (A) Crystal structure of the  Xenopus  nucleosome (PDB 1AOI) illustrating the location and type of fluorescent dye (Cy5 or AlexaFluor647 – abbreviated as A647) used to label histones. Histones are color-coded (H2A – green, H2B – grey, H3 – blue and H4 – magenta) and the two chains of the same histone type can be distinguished by different color shading. For clarity, only one of the two histones of the same type is marked and labelled. (B) SDS-PAGE analysis of wild-type (WT) and fluorescently-labelled histones and histone octamers. Top panel shows Coomassie Brilliant Blue (CBB) staining whereas bottom panel illustrates fluorescence signal of histones labelled with Cy5 or AlexaFluor647. (C) Electrophoretic mobility shift assay (EMSA) for WT and fluorescently-labelled nucleosomes reconstituted on  λ  DNA. Left panel shows SYBR Gold staining of the DNA (magenta), central panel shows Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panel is the composite of both detection modes. Naked  λ  DNA (∼48.5 kbp, first lane) migrates through 0.5 % agarose faster than nucleosomal  λ  templates, containing either WT or fluorescently-labelled histones. (D) Native micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on  λ  DNA. MNase preferentially digests unprotected DNA in linker regions between nucleosomes (see also panel F). Products of MNase digest were resolved in 1.5 % agarose under native conditions revealing intact mono- and di-nucleosomes for nucleosomal templates and complete digest of naked  λ  DNA (first lane). Signal detection as in panel C. (E) Denaturing micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on  λ  DNA. Here, products of MNase digest were first deproteinated with proteinase K (see also panel F) in the presence of SDS and then resolved in 1.5 % agarose, yielding DNA fragments protected by mono-(∼150 bp band) and di-nucleosomes (∼300 bp band) for nucleosomal templates, and short (
    Figure Legend Snippet: Assembly of fluorescent nucleosomes on λ DNA. (A) Crystal structure of the Xenopus nucleosome (PDB 1AOI) illustrating the location and type of fluorescent dye (Cy5 or AlexaFluor647 – abbreviated as A647) used to label histones. Histones are color-coded (H2A – green, H2B – grey, H3 – blue and H4 – magenta) and the two chains of the same histone type can be distinguished by different color shading. For clarity, only one of the two histones of the same type is marked and labelled. (B) SDS-PAGE analysis of wild-type (WT) and fluorescently-labelled histones and histone octamers. Top panel shows Coomassie Brilliant Blue (CBB) staining whereas bottom panel illustrates fluorescence signal of histones labelled with Cy5 or AlexaFluor647. (C) Electrophoretic mobility shift assay (EMSA) for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Left panel shows SYBR Gold staining of the DNA (magenta), central panel shows Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panel is the composite of both detection modes. Naked λ DNA (∼48.5 kbp, first lane) migrates through 0.5 % agarose faster than nucleosomal λ templates, containing either WT or fluorescently-labelled histones. (D) Native micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. MNase preferentially digests unprotected DNA in linker regions between nucleosomes (see also panel F). Products of MNase digest were resolved in 1.5 % agarose under native conditions revealing intact mono- and di-nucleosomes for nucleosomal templates and complete digest of naked λ DNA (first lane). Signal detection as in panel C. (E) Denaturing micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Here, products of MNase digest were first deproteinated with proteinase K (see also panel F) in the presence of SDS and then resolved in 1.5 % agarose, yielding DNA fragments protected by mono-(∼150 bp band) and di-nucleosomes (∼300 bp band) for nucleosomal templates, and short (

    Techniques Used: SDS Page, Staining, Fluorescence, Electrophoretic Mobility Shift Assay

    2) Product Images from "Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction"

    Article Title: Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction

    Journal: Journal of Virology

    doi:

    Comparison of transgene expression and vector copy number per cell in the liver between mice partially hepatectomized after rAAV administration and control mice without hepatectomy. Sixteen adult C57BL/6 mice were injected with rAAV-EF1α-FIX. Three mice were partially hepatectomized on days 3, 10, 17, and 24 and sacrificed 7 days after the hepatectomy. hFIX levels in mouse plasma were determined by an enzyme-linked immunosorbent assay, and the vector copy number per diploid amount of DNA in livers was determined by Southern blot analysis. DNA signals represent all double-stranded vector sequences (see Materials and Methods). Error bars represent standard deviation. The number above each bar indicates the number of mouse samples analyzed. PHx, partial hepatectomy.
    Figure Legend Snippet: Comparison of transgene expression and vector copy number per cell in the liver between mice partially hepatectomized after rAAV administration and control mice without hepatectomy. Sixteen adult C57BL/6 mice were injected with rAAV-EF1α-FIX. Three mice were partially hepatectomized on days 3, 10, 17, and 24 and sacrificed 7 days after the hepatectomy. hFIX levels in mouse plasma were determined by an enzyme-linked immunosorbent assay, and the vector copy number per diploid amount of DNA in livers was determined by Southern blot analysis. DNA signals represent all double-stranded vector sequences (see Materials and Methods). Error bars represent standard deviation. The number above each bar indicates the number of mouse samples analyzed. PHx, partial hepatectomy.

    Techniques Used: Expressing, Plasmid Preparation, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Southern Blot, Standard Deviation

    FISH from mice transduced in vivo with rAAV-FIX and rAAV-TH vectors. C57BL/6-scid mice were infused with 1.0 × 10 11 particles of each rAAV-FIX and rAAV-TH vectors. At 5 weeks later, mouse interphase nuclei from freshly isolated primary hepatocytes were subjected to FISH with fluorescence-labeled rAAV-FIX and/or rAAV-TH probes. Nuclei contained only rAAV-FIX signal (A), only rAAV-TH signal (B), separate rAAV-FIX and rAAV-TH signals (C), or overlapping rAAV-FIX and rAAV-TH signals (D). The discrete red hybridization signals were produced by hybridizing with the rAAV-FIX probe, the green signals were produced by hybridizing with the rAAV-TH probe, and the yellow signal was generated from overlapping red and green signals. Rare cells contained two red or two green signals (not shown). Because some hepatocytes undergo DNA synthesis without nuclear or cell division, it is not possible to determine whether these doubly labeled single-color signals represent two independent events or DNA synthesis after transduction; these were counted as single red or green positive cells.
    Figure Legend Snippet: FISH from mice transduced in vivo with rAAV-FIX and rAAV-TH vectors. C57BL/6-scid mice were infused with 1.0 × 10 11 particles of each rAAV-FIX and rAAV-TH vectors. At 5 weeks later, mouse interphase nuclei from freshly isolated primary hepatocytes were subjected to FISH with fluorescence-labeled rAAV-FIX and/or rAAV-TH probes. Nuclei contained only rAAV-FIX signal (A), only rAAV-TH signal (B), separate rAAV-FIX and rAAV-TH signals (C), or overlapping rAAV-FIX and rAAV-TH signals (D). The discrete red hybridization signals were produced by hybridizing with the rAAV-FIX probe, the green signals were produced by hybridizing with the rAAV-TH probe, and the yellow signal was generated from overlapping red and green signals. Rare cells contained two red or two green signals (not shown). Because some hepatocytes undergo DNA synthesis without nuclear or cell division, it is not possible to determine whether these doubly labeled single-color signals represent two independent events or DNA synthesis after transduction; these were counted as single red or green positive cells.

    Techniques Used: Fluorescence In Situ Hybridization, Mouse Assay, In Vivo, Isolation, Fluorescence, Labeling, Hybridization, Produced, Generated, DNA Synthesis, Transduction

    3) Product Images from "Male meiosis, heterochromatin characterization and chromosomal location of rDNA in Microtomus lunifer (Berg, 1900) (Hemiptera: Reduviidae: Hammacerinae)"

    Article Title: Male meiosis, heterochromatin characterization and chromosomal location of rDNA in Microtomus lunifer (Berg, 1900) (Hemiptera: Reduviidae: Hammacerinae)

    Journal: Comparative Cytogenetics

    doi: 10.3897/compcytogen.v5i1.1143

    Microtomus lunifer . Fluorescent in situ hybridization with an 18S rDNA probe. a Spermatogonial prometaphase b Diplotene c Diakinesis d–e Metaphase I f–g Metaphase II. Hybridization signals in red. Chromosomes are counterstained with DAPI (blue). Arrows: sex chromosomes. White arrowheads: smallest autosomal pair. Bar = 10 µm
    Figure Legend Snippet: Microtomus lunifer . Fluorescent in situ hybridization with an 18S rDNA probe. a Spermatogonial prometaphase b Diplotene c Diakinesis d–e Metaphase I f–g Metaphase II. Hybridization signals in red. Chromosomes are counterstained with DAPI (blue). Arrows: sex chromosomes. White arrowheads: smallest autosomal pair. Bar = 10 µm

    Techniques Used: In Situ Hybridization, Hybridization

    4) Product Images from "The significance of cytogenetics for the study of karyotype evolution and taxonomy of water bugs (Heteroptera, Belostomatidae) native to Argentina"

    Article Title: The significance of cytogenetics for the study of karyotype evolution and taxonomy of water bugs (Heteroptera, Belostomatidae) native to Argentina

    Journal: Comparative Cytogenetics

    doi: 10.3897/CompCytogen.v7i2.4462

    Location of rDNA genes in chromosomes of Belostoma dentatum ( a–d ), Belostoma elongatum ( e, f ) and Belostoma gestroi ( g, h ) by FISH with 18S rDNA probes (red signals, arrows). Chromosomes were counterstained with DAPI (blue). a Spermatogonial anaphase (2n = 29 = 26 + X 1 X 2 Y) b Pachytene c Diffuse stage d Diakinesis e Spermatogonial metaphase and diakinesis f Metaphase I g Spermatogonial metaphase (2n = 29 = 26 + X 1 X 2 Y) h Diakinesis-Metaphase I. Arrowheads show sex chromosomes. Bar = 10 µm.
    Figure Legend Snippet: Location of rDNA genes in chromosomes of Belostoma dentatum ( a–d ), Belostoma elongatum ( e, f ) and Belostoma gestroi ( g, h ) by FISH with 18S rDNA probes (red signals, arrows). Chromosomes were counterstained with DAPI (blue). a Spermatogonial anaphase (2n = 29 = 26 + X 1 X 2 Y) b Pachytene c Diffuse stage d Diakinesis e Spermatogonial metaphase and diakinesis f Metaphase I g Spermatogonial metaphase (2n = 29 = 26 + X 1 X 2 Y) h Diakinesis-Metaphase I. Arrowheads show sex chromosomes. Bar = 10 µm.

    Techniques Used: Fluorescence In Situ Hybridization

    5) Product Images from "A streamlined tethered chromosome conformation capture protocol"

    Article Title: A streamlined tethered chromosome conformation capture protocol

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2596-3

    Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing biotin-14-dCTP (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline
    Figure Legend Snippet: Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing biotin-14-dCTP (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline

    Techniques Used: Sequencing, Magnetic Beads, Purification, Ligation, Generated, Polymerase Chain Reaction, Amplification

    6) Product Images from "Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction"

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2019.00401

    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.
    Figure Legend Snippet: Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Techniques Used: Labeling, Amplification

    Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).
    Figure Legend Snippet: Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Techniques Used:

    7) Product Images from "Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase"

    Article Title: Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl1015

    Experimental scheme and oligonucleotide sequences. ( A ) Principle behind the joining reaction. By exposing complementary sequences, vaccinia DNA polymerase promotes duplex joint formation. ( B ) Sequences of the oligonucleotide substrates used in this paper. Homologous sequences are shown in boldface. The nucleotides shown in lower case are incorporated post-synthetically using reverse transcriptase, cidofovir diphosphate or N 4 -modified biotin-14-dCTP (‘x’), and dATP or dTTP. Note that the primer–template design, in FC3, FC4 and FC5, prevents extension from the other 3′ end by this fill-in reaction. The 32 P label is indicated with an asterisk (‘*’).
    Figure Legend Snippet: Experimental scheme and oligonucleotide sequences. ( A ) Principle behind the joining reaction. By exposing complementary sequences, vaccinia DNA polymerase promotes duplex joint formation. ( B ) Sequences of the oligonucleotide substrates used in this paper. Homologous sequences are shown in boldface. The nucleotides shown in lower case are incorporated post-synthetically using reverse transcriptase, cidofovir diphosphate or N 4 -modified biotin-14-dCTP (‘x’), and dATP or dTTP. Note that the primer–template design, in FC3, FC4 and FC5, prevents extension from the other 3′ end by this fill-in reaction. The 32 P label is indicated with an asterisk (‘*’).

    Techniques Used: Modification

    8) Product Images from "Direct Lentiviral-Cyclooxygenase 2 Application to the Tendon-Bone Interface Promotes Osteointegration and Enhances Return of the Pull-Out Tensile Strength of the Tendon Graft in a Rat Model of Biceps Tenodesis"

    Article Title: Direct Lentiviral-Cyclooxygenase 2 Application to the Tendon-Bone Interface Promotes Osteointegration and Enhances Return of the Pull-Out Tensile Strength of the Tendon Graft in a Rat Model of Biceps Tenodesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098004

    Histology of serial sections of the biceps tendon graft of a representative rat treated with LV- COX2 for 8 weeks. The tenodesis shoulder receiving LV- COX2 vector for 8 weeks was de-mineralized and serially sectioned (5 µm slides from the top) at an orientation perpendicular to the direction of the pin insertion. A and B: Slide 22 of areas around the bony tunnel stained with Toluidine blue (A at 20X, and B at 100 X of the boxed area of panel A); C-F are 100X of the boxed area of panel A in various serial sections (slides 16, 21, 25, and 30, respectively) and each was stained H E. G: a serial section was stained immunohistochemically for type II collagen (stained in brownish color). P: pin hole; S: suture hole; T: tendon; Ch: cartilage; FC: fibrocartilage; and B: bone. The serial sections from panel C to F show that the tendon graft has undergone histological transition from cartilage to fibrocartilage and then to bone. Scale bar in A = 500 µm; and scale bars in B to G = 100 µm.
    Figure Legend Snippet: Histology of serial sections of the biceps tendon graft of a representative rat treated with LV- COX2 for 8 weeks. The tenodesis shoulder receiving LV- COX2 vector for 8 weeks was de-mineralized and serially sectioned (5 µm slides from the top) at an orientation perpendicular to the direction of the pin insertion. A and B: Slide 22 of areas around the bony tunnel stained with Toluidine blue (A at 20X, and B at 100 X of the boxed area of panel A); C-F are 100X of the boxed area of panel A in various serial sections (slides 16, 21, 25, and 30, respectively) and each was stained H E. G: a serial section was stained immunohistochemically for type II collagen (stained in brownish color). P: pin hole; S: suture hole; T: tendon; Ch: cartilage; FC: fibrocartilage; and B: bone. The serial sections from panel C to F show that the tendon graft has undergone histological transition from cartilage to fibrocartilage and then to bone. Scale bar in A = 500 µm; and scale bars in B to G = 100 µm.

    Techniques Used: Plasmid Preparation, Staining

    Quantification of newly formed trabecular bone area at the interface between the pin and bone surface in the bone tunnel of LV- COX2 - or LV- βgal -treated rats after 5 weeks of healing. The area of the newly formed bone was measured with the Osteometric system. Results are shown as mean ±S.D. (n = 6 per test group).
    Figure Legend Snippet: Quantification of newly formed trabecular bone area at the interface between the pin and bone surface in the bone tunnel of LV- COX2 - or LV- βgal -treated rats after 5 weeks of healing. The area of the newly formed bone was measured with the Osteometric system. Results are shown as mean ±S.D. (n = 6 per test group).

    Techniques Used:

    Comparison of the pull-out tensile strength of healing LHB tendon grafts of eight LV-Cox2-treated tenodesis shoulders with that of healing grafts of thirteen LV- βgal -treated control tenodesis shoulders after 5 weeks of healing. The return of pull-out tensile strength of the healing biceps tendon was shown as the relative ratio of the pull-out tensile strength of the operated right shoulders against the pull-out tensile strength of the intact biceps tendon of the left shoulder (i.e., right/left ratio). Results are shown as mean ±S.D. and statistical significance was determined with one-tailed Student's t-test.
    Figure Legend Snippet: Comparison of the pull-out tensile strength of healing LHB tendon grafts of eight LV-Cox2-treated tenodesis shoulders with that of healing grafts of thirteen LV- βgal -treated control tenodesis shoulders after 5 weeks of healing. The return of pull-out tensile strength of the healing biceps tendon was shown as the relative ratio of the pull-out tensile strength of the operated right shoulders against the pull-out tensile strength of the intact biceps tendon of the left shoulder (i.e., right/left ratio). Results are shown as mean ±S.D. and statistical significance was determined with one-tailed Student's t-test.

    Techniques Used: One-tailed Test

    In situ hybridization analyses of human COX2 -expressing cells at the tendon-bone interface of LV- βgal -treated control (A) or LV- COX2 -treated (B and C) rat shoulders. The tenodesis humerus of LV- βgal -treated or LV- COX2 -treated rats were harvested after three weeks of treatment and were each de-mineralized and sectioned at an orientation perpendicular to the direction of the pin insertion. The attachment site of the tendon is beyond the upper right hand corner of each panel. A mixture of three oligonucleotide probes for human COX2 gene were applied to LV- βgal (control)-treated (A) or LV- COX2 -treated (B and C) thin bone sections corresponding to the tendon-bone interface within the bony tunnel. Human COX2 -expressing cells were seen only in the LV- COX2 -treated (B), but not the LV- βgal -treated control sections (A). In panel B, arrows point to the human COX2 -expressing cells. To assist identification of the human COX2 -expressing cell loci, the area corresponding to the human COX2 -expressing cells locus in panel B was enlarged 2.5-time and was shown in panel C. Scale bars = 100 µm.
    Figure Legend Snippet: In situ hybridization analyses of human COX2 -expressing cells at the tendon-bone interface of LV- βgal -treated control (A) or LV- COX2 -treated (B and C) rat shoulders. The tenodesis humerus of LV- βgal -treated or LV- COX2 -treated rats were harvested after three weeks of treatment and were each de-mineralized and sectioned at an orientation perpendicular to the direction of the pin insertion. The attachment site of the tendon is beyond the upper right hand corner of each panel. A mixture of three oligonucleotide probes for human COX2 gene were applied to LV- βgal (control)-treated (A) or LV- COX2 -treated (B and C) thin bone sections corresponding to the tendon-bone interface within the bony tunnel. Human COX2 -expressing cells were seen only in the LV- COX2 -treated (B), but not the LV- βgal -treated control sections (A). In panel B, arrows point to the human COX2 -expressing cells. To assist identification of the human COX2 -expressing cell loci, the area corresponding to the human COX2 -expressing cells locus in panel B was enlarged 2.5-time and was shown in panel C. Scale bars = 100 µm.

    Techniques Used: In Situ Hybridization, Expressing

    Histological staining of the biceps graft of rats treated with the control LV- βgal vector (A E: 20X) or with the LV- COX2 vector (B F: 20X, C and D: 100X of the two boxed areas in B, G: 100X of the boxed area in F) for 3 weeks (A–D) and 8 weeks (E–G), respectively. The tenodesis humerus of a representative rat receiving the LV- βgal control vector (A E) or the LV- COX2 vector (B, C, D, F, and G) was de-mineralized and sectioned at an orientation perpendicular to the direction of the pin insertion. The slice corresponding to the pin-bone tunnel intersection was stained with light green and Safranin-Orange. Cartilage (chondrocytes) areas are stained red. S: suture hole; P: pin hole; Ch: cartilage; Tb: trabecular bone; and B: bone. The arrows in B show various foci of neo-cartilage formation at the interface between the tendon graft and the bone surface of the bony tunnel. The blue arrow in G shows the site of neo-cartilage formation where columns of chondrocytes were organized in parallel to the tendon fibers, a characteristic suggestive of being fibrocartilage. Panel H and I show two representative areas of the immunohistochemical staining for type II collagen (stained in brownish color) on a serial section (100X). Scale bars = 100 µm.
    Figure Legend Snippet: Histological staining of the biceps graft of rats treated with the control LV- βgal vector (A E: 20X) or with the LV- COX2 vector (B F: 20X, C and D: 100X of the two boxed areas in B, G: 100X of the boxed area in F) for 3 weeks (A–D) and 8 weeks (E–G), respectively. The tenodesis humerus of a representative rat receiving the LV- βgal control vector (A E) or the LV- COX2 vector (B, C, D, F, and G) was de-mineralized and sectioned at an orientation perpendicular to the direction of the pin insertion. The slice corresponding to the pin-bone tunnel intersection was stained with light green and Safranin-Orange. Cartilage (chondrocytes) areas are stained red. S: suture hole; P: pin hole; Ch: cartilage; Tb: trabecular bone; and B: bone. The arrows in B show various foci of neo-cartilage formation at the interface between the tendon graft and the bone surface of the bony tunnel. The blue arrow in G shows the site of neo-cartilage formation where columns of chondrocytes were organized in parallel to the tendon fibers, a characteristic suggestive of being fibrocartilage. Panel H and I show two representative areas of the immunohistochemical staining for type II collagen (stained in brownish color) on a serial section (100X). Scale bars = 100 µm.

    Techniques Used: Staining, Plasmid Preparation, Immunohistochemistry

    A schematic representation of the structure of expression plasmids used to produce the third-generation self-inactivating LV- COX2 vector. The transfer expression construct (pLV- COX2 plasmid) used the cytomegalovirus (CMV) promoter to drive expression of a human COX2 gene that had been modified by removing the 3′-untranslated region (UTR) that contains multiple copies of the AUUA-rich element to increase the mRNA stability and also by replacing the native Kozak sequence with an enhanced Kozak sequence to improve protein translation (22). The expression of gag , pol , and RRE lentiviral genes on the pLV-GP plasmid, the Rev gene on the pLV-Rev plasmid, and the heterologus VSV-G envelope gene on the pVSV-G plasmid was driven by the CMV promoter. The transfer construct did not contain the wild-type copy of the LV LTR promoter (thereby self-inactivating). The 5′-LTR was chimeric, containing a CMV promoter replacing the U3 region to rescue transcriptional dependence on the tat lentiviral gene. The 3′-LTR (ΔLTR) contained a deletion through the U3 region that renders it transcriptionally inactive.
    Figure Legend Snippet: A schematic representation of the structure of expression plasmids used to produce the third-generation self-inactivating LV- COX2 vector. The transfer expression construct (pLV- COX2 plasmid) used the cytomegalovirus (CMV) promoter to drive expression of a human COX2 gene that had been modified by removing the 3′-untranslated region (UTR) that contains multiple copies of the AUUA-rich element to increase the mRNA stability and also by replacing the native Kozak sequence with an enhanced Kozak sequence to improve protein translation (22). The expression of gag , pol , and RRE lentiviral genes on the pLV-GP plasmid, the Rev gene on the pLV-Rev plasmid, and the heterologus VSV-G envelope gene on the pVSV-G plasmid was driven by the CMV promoter. The transfer construct did not contain the wild-type copy of the LV LTR promoter (thereby self-inactivating). The 5′-LTR was chimeric, containing a CMV promoter replacing the U3 region to rescue transcriptional dependence on the tat lentiviral gene. The 3′-LTR (ΔLTR) contained a deletion through the U3 region that renders it transcriptionally inactive.

    Techniques Used: Expressing, Plasmid Preparation, Construct, Modification, Sequencing

    Comparison of the effect of the LV- COX2 in vivo gene transfer strategy with that of the LV- BMP4 in vivo gene transfer strategy on de novo angiogenesis at the tendon-bone interface within the bony tunnel. To identify blood vessels, thin sections of humerus containing the bony tunnel and the tendon graft of animals treated with LV- COX2 , LV- BMP4 , or LV- βgal control vector for either 3 weeks or 5 weeks were stained immunohistologically for vWF (brownish color) using a specific anti-rat-vWF antibody. Scale bars = 100 µm. Bottom shows the quantification of the area stained for vWF per area soft tissue around the pinhole. Four individual samples each were examined for LV- COX2 and LV- βgal (control) therapy at three weeks post-procedure. Three different areas of soft tissue were examined in each section. Statistical analysis was performed by two-tailed Student's t-Test.
    Figure Legend Snippet: Comparison of the effect of the LV- COX2 in vivo gene transfer strategy with that of the LV- BMP4 in vivo gene transfer strategy on de novo angiogenesis at the tendon-bone interface within the bony tunnel. To identify blood vessels, thin sections of humerus containing the bony tunnel and the tendon graft of animals treated with LV- COX2 , LV- BMP4 , or LV- βgal control vector for either 3 weeks or 5 weeks were stained immunohistologically for vWF (brownish color) using a specific anti-rat-vWF antibody. Scale bars = 100 µm. Bottom shows the quantification of the area stained for vWF per area soft tissue around the pinhole. Four individual samples each were examined for LV- COX2 and LV- βgal (control) therapy at three weeks post-procedure. Three different areas of soft tissue were examined in each section. Statistical analysis was performed by two-tailed Student's t-Test.

    Techniques Used: In Vivo, Plasmid Preparation, Staining, Two Tailed Test

    9) Product Images from "Chromosome Mapping of Repetitive Sequences in Rachycentron canadum (Perciformes: Rachycentridae): Implications for Karyotypic Evolution and Perspectives for Biotechnological Uses"

    Article Title: Chromosome Mapping of Repetitive Sequences in Rachycentron canadum (Perciformes: Rachycentridae): Implications for Karyotypic Evolution and Perspectives for Biotechnological Uses

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2011/218231

    Karyotypes of Rachycentron canadum. Conventional staining (a) highlighting the Ag-NOR sites in chromosome 2; (b) C-banding, highlighting the NORs as CMA 3 + /DAPI − heterochromatic regions; (c) replication bands, showing 18S (pair 2) and 5S (pairs 3 and 13) rDNA sites with early replication; (d) double - FISH with 18S (pink) and 5S (green) rDNA probes, showing the location of 18S rDNA sites in pair 2 and of 5S rDNA sites in pairs 3 and 13; (e) FISH with (TTAGGG) n sequences showing the location of telomeric sites in the chromosomes (orange). Bar = 5 μ m.
    Figure Legend Snippet: Karyotypes of Rachycentron canadum. Conventional staining (a) highlighting the Ag-NOR sites in chromosome 2; (b) C-banding, highlighting the NORs as CMA 3 + /DAPI − heterochromatic regions; (c) replication bands, showing 18S (pair 2) and 5S (pairs 3 and 13) rDNA sites with early replication; (d) double - FISH with 18S (pink) and 5S (green) rDNA probes, showing the location of 18S rDNA sites in pair 2 and of 5S rDNA sites in pairs 3 and 13; (e) FISH with (TTAGGG) n sequences showing the location of telomeric sites in the chromosomes (orange). Bar = 5 μ m.

    Techniques Used: Staining, Fluorescence In Situ Hybridization

    10) Product Images from "Studies in two allopatric populations of Hypostomusaffinis (Steindachner, 1877): the role of mapping the ribosomal genes to understand the chromosome evolution of the group"

    Article Title: Studies in two allopatric populations of Hypostomusaffinis (Steindachner, 1877): the role of mapping the ribosomal genes to understand the chromosome evolution of the group

    Journal: Comparative Cytogenetics

    doi: 10.3897/CompCytogen.v12i1.22052

    Karyotypes found for the populations of Cunha/SP ( A ) and Itaocara/RJ ( B ). In the boxes are the phenotypes for C banding, Ag- NORs and FISH with 5S and 18S rDNA probes.
    Figure Legend Snippet: Karyotypes found for the populations of Cunha/SP ( A ) and Itaocara/RJ ( B ). In the boxes are the phenotypes for C banding, Ag- NORs and FISH with 5S and 18S rDNA probes.

    Techniques Used: Fluorescence In Situ Hybridization

    11) Product Images from "Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification"

    Article Title: Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification

    Journal: Molecular Cytogenetics

    doi: 10.1186/s13039-017-0338-0

    Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    12) Product Images from "The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops "

    Article Title: The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

    Journal: The Journal of Cell Biology

    doi:

    SATB1-binding sites are localized to the base of chromatin loops. In situ hybridization with Jurkat nuclei ( top ), nuclear halo ( middle ), and nuclear matrix ( bottom ) was performed using in vivo SATB1-binding sequences, SBS-2, SBS-3, and SBS-11, and control DNA, pTP18 (for U2 snRNA) and pH5SB (for the 5S rRNA gene), as DNA probes. These probes were labeled with biotin-11-dATP. Total DNA was stained with propidium iodide ( red ), and specific sequence hybridization was detected with FITC-conjugated extravidin ( yellow-green ). Since most of the genomic DNA has been removed from the nuclear matrix preparations, no propidium iodide staining was detectable in the nuclear matrix. In situ hybridization signals to the 5S rRNA within the distended chromatin halo are indicated by a white arrow.
    Figure Legend Snippet: SATB1-binding sites are localized to the base of chromatin loops. In situ hybridization with Jurkat nuclei ( top ), nuclear halo ( middle ), and nuclear matrix ( bottom ) was performed using in vivo SATB1-binding sequences, SBS-2, SBS-3, and SBS-11, and control DNA, pTP18 (for U2 snRNA) and pH5SB (for the 5S rRNA gene), as DNA probes. These probes were labeled with biotin-11-dATP. Total DNA was stained with propidium iodide ( red ), and specific sequence hybridization was detected with FITC-conjugated extravidin ( yellow-green ). Since most of the genomic DNA has been removed from the nuclear matrix preparations, no propidium iodide staining was detectable in the nuclear matrix. In situ hybridization signals to the 5S rRNA within the distended chromatin halo are indicated by a white arrow.

    Techniques Used: Binding Assay, In Situ Hybridization, In Vivo, Labeling, Staining, Sequencing, Hybridization

    13) Product Images from "Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification"

    Article Title: Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification

    Journal: Molecular Cytogenetics

    doi: 10.1186/s13039-017-0338-0

    Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    14) Product Images from "Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don"

    Article Title: Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don

    Journal: AoB Plants

    doi: 10.1093/aobpla/pls002

    Cytogenetic analyses of C. roseus on mitotic spreads prepared from root tip cells . (A–C) Mitotic spreads of C. roseus stained with silver nitrate. (A) Prophase revealing a single nucleolus. (B) Prometaphase revealing a single pair of homologous chromosomes associated to the nucleolus. (C) Prometaphase with an already disorganized nucleolus and with the NORs restricted to a single chromosome pair. (D) Fluorescent staining with AD and DAPI of a prometaphase. (E) C-banding and staining with PI and DAPI of a prometaphase, highlighting the NORs in red. (F) Fluorescence in situ hybridization of rDNA of a metaphase. The probe (pTa71) was labelled with biotin-14-dATP and detected with fluorescein-avidin D, the chromosomes were counterstained with DAPI and the NORs are highlighted in green. Arrow indicates chromosome 8. (G) Catharanthus roseus karyotype. Chromosomes are aligned by the centromeric region and are ordered from 1 to 8, from the larger to the smaller, according to the morphometric analysis. (H) Ideogram of C. roseus chromosomes. Centromeres, constrictions, bands and NORs are depicted in the picture; the small arm of chromosome 8 is represented by a dashed line since it is usually not visible. Scale bars = 5 μm.
    Figure Legend Snippet: Cytogenetic analyses of C. roseus on mitotic spreads prepared from root tip cells . (A–C) Mitotic spreads of C. roseus stained with silver nitrate. (A) Prophase revealing a single nucleolus. (B) Prometaphase revealing a single pair of homologous chromosomes associated to the nucleolus. (C) Prometaphase with an already disorganized nucleolus and with the NORs restricted to a single chromosome pair. (D) Fluorescent staining with AD and DAPI of a prometaphase. (E) C-banding and staining with PI and DAPI of a prometaphase, highlighting the NORs in red. (F) Fluorescence in situ hybridization of rDNA of a metaphase. The probe (pTa71) was labelled with biotin-14-dATP and detected with fluorescein-avidin D, the chromosomes were counterstained with DAPI and the NORs are highlighted in green. Arrow indicates chromosome 8. (G) Catharanthus roseus karyotype. Chromosomes are aligned by the centromeric region and are ordered from 1 to 8, from the larger to the smaller, according to the morphometric analysis. (H) Ideogram of C. roseus chromosomes. Centromeres, constrictions, bands and NORs are depicted in the picture; the small arm of chromosome 8 is represented by a dashed line since it is usually not visible. Scale bars = 5 μm.

    Techniques Used: Staining, Fluorescence, In Situ Hybridization, Avidin-Biotin Assay

    15) Product Images from "A streamlined tethered chromosome conformation capture protocol"

    Article Title: A streamlined tethered chromosome conformation capture protocol

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2596-3

    Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing biotin-14-dCTP (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline
    Figure Legend Snippet: Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing biotin-14-dCTP (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline

    Techniques Used: Sequencing, Magnetic Beads, Purification, Ligation, Generated, Polymerase Chain Reaction, Amplification

    16) Product Images from "Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification"

    Article Title: Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification

    Journal: Molecular Cytogenetics

    doi: 10.1186/s13039-017-0338-0

    Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes.
    Figure Legend Snippet: Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes.

    Techniques Used:

    Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes.
    Figure Legend Snippet: Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes.

    Techniques Used:

    Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale
    Figure Legend Snippet: Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale

    Techniques Used:

    17) Product Images from "The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair"

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw274

    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    Figure Legend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.
    Figure Legend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    18) Product Images from "Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification"

    Article Title: Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification

    Journal: Molecular Cytogenetics

    doi: 10.1186/s13039-017-0338-0

    Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Larimichthys crocea mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Larimichthys polyactis mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm
    Figure Legend Snippet: Metaphase plates of Nibea albiflora mapped with different repeated DNAs . 5S rDNA (green), 18S rDNA, di-and trinucleotide microsatellites, Rex 1 and U2 snRNA (red) as probes. The chromosomes bearing 18S rDNA sites are shown in enlarged forms boxes. Scale bar = 5 μm

    Techniques Used:

    19) Product Images from "Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry"

    Article Title: Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

    Journal: Nucleic Acids Research

    doi:

    Chromosome paints generated from chromosomes sorted on the basis of the intensity of bound S-PA-F polyamide and HO. The chromosomes used to produce the paints in (A)–(D) correspond to the sort windows a–d in Figure 3A. In each case, 2000 chromosomes were sorted, amplified and biotinylated by DOP-PCR, hybridized to metaphase chromosomes and detected with fluorescein–avidin. The fluorescein-bright chromosomes in sort window a yield a chromosome 9-specific paint ( A ); the fluorescein-dim chromosomes with similar HO fluorescence intensities (sort window b) paint chromosomes 10, 11 and 12 ( B ). The fluorescein-bright chromosomes in sort window c yield a chromosome Y-specific paint (the long arm is unlabeled due to the suppression of the hybridization of highly repetitive sequences in the paint probe) ( C ); the fluorescein-dim chromosomes with a similar HO fluorescence intensity (sort window d) paint chromosome 18 ( D ). All metaphases were counterstained with DAPI.
    Figure Legend Snippet: Chromosome paints generated from chromosomes sorted on the basis of the intensity of bound S-PA-F polyamide and HO. The chromosomes used to produce the paints in (A)–(D) correspond to the sort windows a–d in Figure 3A. In each case, 2000 chromosomes were sorted, amplified and biotinylated by DOP-PCR, hybridized to metaphase chromosomes and detected with fluorescein–avidin. The fluorescein-bright chromosomes in sort window a yield a chromosome 9-specific paint ( A ); the fluorescein-dim chromosomes with similar HO fluorescence intensities (sort window b) paint chromosomes 10, 11 and 12 ( B ). The fluorescein-bright chromosomes in sort window c yield a chromosome Y-specific paint (the long arm is unlabeled due to the suppression of the hybridization of highly repetitive sequences in the paint probe) ( C ); the fluorescein-dim chromosomes with a similar HO fluorescence intensity (sort window d) paint chromosome 18 ( D ). All metaphases were counterstained with DAPI.

    Techniques Used: Generated, Amplification, Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Avidin-Biotin Assay, Fluorescence, Hybridization

    20) Product Images from "Human T-Lymphotropic Virus Type 1 p30II Regulates Gene Transcription by Binding CREB Binding Protein/p300"

    Article Title: Human T-Lymphotropic Virus Type 1 p30II Regulates Gene Transcription by Binding CREB Binding Protein/p300

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.20.9885-9895.2001

    p30 II inhibits the assembly of Tax-p300-CREB multiprotein complexes on TRE oligonucleotides. Exogenous Tax, p300, and p30 II -HA were introduced into 293T cells by transfection using expression plasmids. At 48 h posttransfection, nuclear lysates of transfected cells were incubated with the biotin-labeled TRE (HTLV-1 21-bp repeat) DNA probe as described in Materials and Methods. Streptavidin-agarose was used to isolate bound components. After extensive washing, bound proteins (right panel) were resolved using SDS-PAGE (4 to 20% gradient acrylamide) gels and detected by Western immunoblot assay. In parallel, equal amounts of nuclear lysates used as input were examined by Western immunoblot assay to determine protein expression levels of each component in transfected cells (left panel).
    Figure Legend Snippet: p30 II inhibits the assembly of Tax-p300-CREB multiprotein complexes on TRE oligonucleotides. Exogenous Tax, p300, and p30 II -HA were introduced into 293T cells by transfection using expression plasmids. At 48 h posttransfection, nuclear lysates of transfected cells were incubated with the biotin-labeled TRE (HTLV-1 21-bp repeat) DNA probe as described in Materials and Methods. Streptavidin-agarose was used to isolate bound components. After extensive washing, bound proteins (right panel) were resolved using SDS-PAGE (4 to 20% gradient acrylamide) gels and detected by Western immunoblot assay. In parallel, equal amounts of nuclear lysates used as input were examined by Western immunoblot assay to determine protein expression levels of each component in transfected cells (left panel).

    Techniques Used: Transfection, Expressing, Incubation, Labeling, SDS Page, Western Blot

    21) Product Images from "Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction"

    Article Title: Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction

    Journal: Journal of Virology

    doi:

    Comparison of transgene expression and vector copy number per cell in the liver between mice partially hepatectomized after rAAV administration and control mice without hepatectomy. Sixteen adult C57BL/6 mice were injected with rAAV-EF1α-FIX. Three mice were partially hepatectomized on days 3, 10, 17, and 24 and sacrificed 7 days after the hepatectomy. hFIX levels in mouse plasma were determined by an enzyme-linked immunosorbent assay, and the vector copy number per diploid amount of DNA in livers was determined by Southern blot analysis. DNA signals represent all double-stranded vector sequences (see Materials and Methods). Error bars represent standard deviation. The number above each bar indicates the number of mouse samples analyzed. PHx, partial hepatectomy.
    Figure Legend Snippet: Comparison of transgene expression and vector copy number per cell in the liver between mice partially hepatectomized after rAAV administration and control mice without hepatectomy. Sixteen adult C57BL/6 mice were injected with rAAV-EF1α-FIX. Three mice were partially hepatectomized on days 3, 10, 17, and 24 and sacrificed 7 days after the hepatectomy. hFIX levels in mouse plasma were determined by an enzyme-linked immunosorbent assay, and the vector copy number per diploid amount of DNA in livers was determined by Southern blot analysis. DNA signals represent all double-stranded vector sequences (see Materials and Methods). Error bars represent standard deviation. The number above each bar indicates the number of mouse samples analyzed. PHx, partial hepatectomy.

    Techniques Used: Expressing, Plasmid Preparation, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Southern Blot, Standard Deviation

    FISH from mice transduced in vivo with rAAV-FIX and rAAV-TH vectors. C57BL/6-scid mice were infused with 1.0 × 10 11 particles of each rAAV-FIX and rAAV-TH vectors. At 5 weeks later, mouse interphase nuclei from freshly isolated primary hepatocytes were subjected to FISH with fluorescence-labeled rAAV-FIX and/or rAAV-TH probes. Nuclei contained only rAAV-FIX signal (A), only rAAV-TH signal (B), separate rAAV-FIX and rAAV-TH signals (C), or overlapping rAAV-FIX and rAAV-TH signals (D). The discrete red hybridization signals were produced by hybridizing with the rAAV-FIX probe, the green signals were produced by hybridizing with the rAAV-TH probe, and the yellow signal was generated from overlapping red and green signals. Rare cells contained two red or two green signals (not shown). Because some hepatocytes undergo DNA synthesis without nuclear or cell division, it is not possible to determine whether these doubly labeled single-color signals represent two independent events or DNA synthesis after transduction; these were counted as single red or green positive cells.
    Figure Legend Snippet: FISH from mice transduced in vivo with rAAV-FIX and rAAV-TH vectors. C57BL/6-scid mice were infused with 1.0 × 10 11 particles of each rAAV-FIX and rAAV-TH vectors. At 5 weeks later, mouse interphase nuclei from freshly isolated primary hepatocytes were subjected to FISH with fluorescence-labeled rAAV-FIX and/or rAAV-TH probes. Nuclei contained only rAAV-FIX signal (A), only rAAV-TH signal (B), separate rAAV-FIX and rAAV-TH signals (C), or overlapping rAAV-FIX and rAAV-TH signals (D). The discrete red hybridization signals were produced by hybridizing with the rAAV-FIX probe, the green signals were produced by hybridizing with the rAAV-TH probe, and the yellow signal was generated from overlapping red and green signals. Rare cells contained two red or two green signals (not shown). Because some hepatocytes undergo DNA synthesis without nuclear or cell division, it is not possible to determine whether these doubly labeled single-color signals represent two independent events or DNA synthesis after transduction; these were counted as single red or green positive cells.

    Techniques Used: Fluorescence In Situ Hybridization, Mouse Assay, In Vivo, Isolation, Fluorescence, Labeling, Hybridization, Produced, Generated, DNA Synthesis, Transduction

    22) Product Images from "Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction"

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2019.00401

    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.
    Figure Legend Snippet: Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Techniques Used: Labeling, Amplification

    Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).
    Figure Legend Snippet: Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Techniques Used:

    23) Product Images from "Comparative study of mitotic chromosomes in two blowflies, Luciliasericata and L.cluvia ( Diptera, Calliphoridae), by C- and G-like banding patterns and rRNA loci, and implications for karyotype evolution"

    Article Title: Comparative study of mitotic chromosomes in two blowflies, Luciliasericata and L.cluvia ( Diptera, Calliphoridae), by C- and G-like banding patterns and rRNA loci, and implications for karyotype evolution

    Journal: Comparative Cytogenetics

    doi: 10.3897/CompCytogen.v9i1.8671

    Location of rDNA genes and Ag-NOR sites on female and male mitotic chromosomes of Lucilia cluvia ( a–b ) and Lucilia sericata ( c–d , e–f ) using FISH with 18S rDNA probes (red signals, arrowheads) and silver impregnation technique. Chromosomes were counterstained with DAPI (blue). X, Y = sex chromosomes. Arrowheads indicate hybridization signals ( a–d ) and Ag-NOR sites ( e–f ) in both sex chromosomes. Bar = 10 μm.
    Figure Legend Snippet: Location of rDNA genes and Ag-NOR sites on female and male mitotic chromosomes of Lucilia cluvia ( a–b ) and Lucilia sericata ( c–d , e–f ) using FISH with 18S rDNA probes (red signals, arrowheads) and silver impregnation technique. Chromosomes were counterstained with DAPI (blue). X, Y = sex chromosomes. Arrowheads indicate hybridization signals ( a–d ) and Ag-NOR sites ( e–f ) in both sex chromosomes. Bar = 10 μm.

    Techniques Used: Fluorescence In Situ Hybridization, Hybridization

    24) Product Images from "Cytogenetics of the Porthole Shovelnose Catfish, Hemisorubim platyrhynchos (Valenciennes, 1840) (Siluriformes, Pimelodidae), a widespread species in South American rivers"

    Article Title: Cytogenetics of the Porthole Shovelnose Catfish, Hemisorubim platyrhynchos (Valenciennes, 1840) (Siluriformes, Pimelodidae), a widespread species in South American rivers

    Journal: Comparative Cytogenetics

    doi: 10.3897/CompCytogen.v7i2.4901

    Metaphases of Hemisorubim platyrhynchos showing sequential Giemsa-AgNO 3 staining ( a, b ) CMA 3 banding ( c ) FISH with 18S rDNA probe ( d ). Arrows indicate the NOR- bearing chromosomes.
    Figure Legend Snippet: Metaphases of Hemisorubim platyrhynchos showing sequential Giemsa-AgNO 3 staining ( a, b ) CMA 3 banding ( c ) FISH with 18S rDNA probe ( d ). Arrows indicate the NOR- bearing chromosomes.

    Techniques Used: Staining, Fluorescence In Situ Hybridization

    25) Product Images from "Tracking the evolution of sex chromosome systems in Melanoplinae grasshoppers through chromosomal mapping of repetitive DNA sequences"

    Article Title: Tracking the evolution of sex chromosome systems in Melanoplinae grasshoppers through chromosomal mapping of repetitive DNA sequences

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-13-167

    C-banding and C 0 t -1 DNA fractions in female mitotic chromosomes. The species names and the types of sex chromosome determination systems are indicated in each figure. Inserts show the locations of C-banding and C 0 t -1 DNA fractions in the neo-sex chromosomes during meiosis. Note the absence of the C 0 t -1 DNA fraction in the neo-Y chromosome of D. lilloanus. Bar = 5 μm.
    Figure Legend Snippet: C-banding and C 0 t -1 DNA fractions in female mitotic chromosomes. The species names and the types of sex chromosome determination systems are indicated in each figure. Inserts show the locations of C-banding and C 0 t -1 DNA fractions in the neo-sex chromosomes during meiosis. Note the absence of the C 0 t -1 DNA fraction in the neo-Y chromosome of D. lilloanus. Bar = 5 μm.

    Techniques Used:

    26) Product Images from "Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes"

    Article Title: Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

    Journal: Genetics and Molecular Biology

    doi: 10.1590/S1415-47572010005000050

    Meiotic cells from Astylus antis : a. metaphase I cell stained with Giemsa, 2n = 8II+Xyp; b. the same cell stained with silver nitrate, showing strong impregnation associated to the sex chromosomes (arrowhead); c. and d. pachytene and metaphase I cells, respectively, hybridized with an 18S rDNA probe, showing an autosomal bivalent with a fluorescent signal (arrows). Bar = 5 μm.
    Figure Legend Snippet: Meiotic cells from Astylus antis : a. metaphase I cell stained with Giemsa, 2n = 8II+Xyp; b. the same cell stained with silver nitrate, showing strong impregnation associated to the sex chromosomes (arrowhead); c. and d. pachytene and metaphase I cells, respectively, hybridized with an 18S rDNA probe, showing an autosomal bivalent with a fluorescent signal (arrows). Bar = 5 μm.

    Techniques Used: Staining

    27) Product Images from "Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation"

    Article Title: Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation

    Journal: Nature genetics

    doi: 10.1038/ng.3935

    Remodeling of the enhancer–promoter contact landscape in terminal differentiation. ( a ) Genomic locus of keratin-family genes. From top to bottom: Hi-C interaction matrices, self-interacting domains (green), and genes induced (red) or repressed (blue) on day 3 or day 6 of epidermal cell differentiation identified by RNA-seq. ( b ) Scatterplot of Hi-C read counts supporting contacts between domain boundaries. Contacts enclosing domains containing differentially expressed genes are highlighted. ( c ) Heat map of CHi-C read count profiles observed for bait HindIII fragments residing within a domain. Each row represents the contact signal from a single promoter, promoters are aligned by distance to the upstream domain boundary, and rows are sorted in order of domain size. ( d ) Heat maps of CHi-C q scores (determined by CHiCAGO) for 1,975 enhancer contacts gained with target promoters during epidermal cell differentiation. Contacts are sorted in order of enhancer–promoter (EP) distance. (edgeR, FDR
    Figure Legend Snippet: Remodeling of the enhancer–promoter contact landscape in terminal differentiation. ( a ) Genomic locus of keratin-family genes. From top to bottom: Hi-C interaction matrices, self-interacting domains (green), and genes induced (red) or repressed (blue) on day 3 or day 6 of epidermal cell differentiation identified by RNA-seq. ( b ) Scatterplot of Hi-C read counts supporting contacts between domain boundaries. Contacts enclosing domains containing differentially expressed genes are highlighted. ( c ) Heat map of CHi-C read count profiles observed for bait HindIII fragments residing within a domain. Each row represents the contact signal from a single promoter, promoters are aligned by distance to the upstream domain boundary, and rows are sorted in order of domain size. ( d ) Heat maps of CHi-C q scores (determined by CHiCAGO) for 1,975 enhancer contacts gained with target promoters during epidermal cell differentiation. Contacts are sorted in order of enhancer–promoter (EP) distance. (edgeR, FDR

    Techniques Used: Hi-C, Cell Differentiation, RNA Sequencing Assay

    28) Product Images from "Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray"

    Article Title: Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000924

    Strategy for amplification and labeling of pathogen-specific target. The one-tube RT-PCR reaction includes three stages: A) Reverse transcription reaction to produce first strand cDNA for viral targets (vRNA); B) Low temperature (56°C), exponential amplification of target and C) High temperature (68°C), linear amplification to product single-strand, biotin-labeled target. Target is labeled by incorporation of biotin-14-dCTP during amplification or by the use of biotinylated reverse primers.
    Figure Legend Snippet: Strategy for amplification and labeling of pathogen-specific target. The one-tube RT-PCR reaction includes three stages: A) Reverse transcription reaction to produce first strand cDNA for viral targets (vRNA); B) Low temperature (56°C), exponential amplification of target and C) High temperature (68°C), linear amplification to product single-strand, biotin-labeled target. Target is labeled by incorporation of biotin-14-dCTP during amplification or by the use of biotinylated reverse primers.

    Techniques Used: Amplification, Labeling, Reverse Transcription Polymerase Chain Reaction

    29) Product Images from "ISWI Remodeling Complexes in Xenopus Egg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B"

    Article Title: ISWI Remodeling Complexes in Xenopus Egg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.01-09-0441

    Effect of XISWI-, XACF1-, or XWSTF-depletion on interphase nuclear functions. (A) Interphase HSS was depleted with control IgG (lane 1), anti-XISWI (lane 2), anti-XACF1 (lane 3), or anti-XWSTF antibody (lane 4). Equal volumes of each HSS were analyzed by immunoblotting with the antibodies indicated. (B) Histone deposition assay. Supercoiled plasmid DNA was incubated with the control HSS (Δmock) or the depleted HSSs (ΔXISWI, ΔXACF1, and ΔXWSTF). Aliquots were taken at the time points indicated, deproteinated, separated on a 1.25% agarose gel, and stained with ethidium bromide. I, supercoiled DNA; Ir, relaxed DNA; II, nicked circular DNA. (C) Nucleosome spacing assay. Sperm chromatin was incubated with control or depleted HSS for 90 min. Each sample was then divided into three aliquots and treated with increasing concentrations of micrococcal nuclease (MNase: 0.36 U/ml, lanes 1, 4, 7, and 10; 1.2 U/ml, lanes 2, 5, 8, and 11; and 3.6 U/ml, lanes 3, 6, 9, and 12). After deproteination, the DNA was separated on a 1.25% agarose gel and stained with ethidium bromide. A 100-base pair ladder was used as a molecular weight marker. The positions of nucleosomal oligomers are indicated. (D) DNA replication assay. Sperm chromatin was incubated with control or depleted LSS in the presence of [α- 32 P]dATP. Aliquots were taken at the time points indicated, deproteinated, precipitated with TCA, and placed on a filter for scintillation counting. The extent of replication is expressed as nanograms of DNA replicated per microliter of extract.
    Figure Legend Snippet: Effect of XISWI-, XACF1-, or XWSTF-depletion on interphase nuclear functions. (A) Interphase HSS was depleted with control IgG (lane 1), anti-XISWI (lane 2), anti-XACF1 (lane 3), or anti-XWSTF antibody (lane 4). Equal volumes of each HSS were analyzed by immunoblotting with the antibodies indicated. (B) Histone deposition assay. Supercoiled plasmid DNA was incubated with the control HSS (Δmock) or the depleted HSSs (ΔXISWI, ΔXACF1, and ΔXWSTF). Aliquots were taken at the time points indicated, deproteinated, separated on a 1.25% agarose gel, and stained with ethidium bromide. I, supercoiled DNA; Ir, relaxed DNA; II, nicked circular DNA. (C) Nucleosome spacing assay. Sperm chromatin was incubated with control or depleted HSS for 90 min. Each sample was then divided into three aliquots and treated with increasing concentrations of micrococcal nuclease (MNase: 0.36 U/ml, lanes 1, 4, 7, and 10; 1.2 U/ml, lanes 2, 5, 8, and 11; and 3.6 U/ml, lanes 3, 6, 9, and 12). After deproteination, the DNA was separated on a 1.25% agarose gel and stained with ethidium bromide. A 100-base pair ladder was used as a molecular weight marker. The positions of nucleosomal oligomers are indicated. (D) DNA replication assay. Sperm chromatin was incubated with control or depleted LSS in the presence of [α- 32 P]dATP. Aliquots were taken at the time points indicated, deproteinated, precipitated with TCA, and placed on a filter for scintillation counting. The extent of replication is expressed as nanograms of DNA replicated per microliter of extract.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker

    30) Product Images from "Presence and integration of HBV DNA in mouse oocytes"

    Article Title: Presence and integration of HBV DNA in mouse oocytes

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v11.i19.2869

    FISH results. A: No signal of HBV DNA integration in the metaphase from group B oocytes. B-D: Clear signals of HBV DNA integration in the metaphases from group A oocytes (arrow).
    Figure Legend Snippet: FISH results. A: No signal of HBV DNA integration in the metaphase from group B oocytes. B-D: Clear signals of HBV DNA integration in the metaphases from group A oocytes (arrow).

    Techniques Used: Fluorescence In Situ Hybridization

    31) Product Images from "Phylogeny and chromosomal diversification in the Dichroplus elongatus species group (Orthoptera, Melanoplinae)"

    Article Title: Phylogeny and chromosomal diversification in the Dichroplus elongatus species group (Orthoptera, Melanoplinae)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172352

    FISH with 18S, 5S rDNA, H3 histone and U2 snDNA probes in meiotic cells from males. The probe and species name are indicated in each figure. Chromosomes with positive signals and the X chromosome are indicated. (A-D) 18S rDNA, (E-H) 5S rDNA, (I-L) H3 histone gene and (M-P) U2 snDNA. Bar = 10 μm.
    Figure Legend Snippet: FISH with 18S, 5S rDNA, H3 histone and U2 snDNA probes in meiotic cells from males. The probe and species name are indicated in each figure. Chromosomes with positive signals and the X chromosome are indicated. (A-D) 18S rDNA, (E-H) 5S rDNA, (I-L) H3 histone gene and (M-P) U2 snDNA. Bar = 10 μm.

    Techniques Used: Fluorescence In Situ Hybridization

    32) Product Images from "Tracking the evolution of sex chromosome systems in Melanoplinae grasshoppers through chromosomal mapping of repetitive DNA sequences"

    Article Title: Tracking the evolution of sex chromosome systems in Melanoplinae grasshoppers through chromosomal mapping of repetitive DNA sequences

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-13-167

    FISH for the histone H3 gene in male meiotic cells. Divergent sex chromosomes, names of species and the chromosomes with hybridization signals are indicated in the figure. Remarkably, the histone H3 gene cluster was found in pericentromeric regions of all chromosomes, except in the neo-Y in D. lilloanus . Bar = 5 μm.
    Figure Legend Snippet: FISH for the histone H3 gene in male meiotic cells. Divergent sex chromosomes, names of species and the chromosomes with hybridization signals are indicated in the figure. Remarkably, the histone H3 gene cluster was found in pericentromeric regions of all chromosomes, except in the neo-Y in D. lilloanus . Bar = 5 μm.

    Techniques Used: Fluorescence In Situ Hybridization, Hybridization

    33) Product Images from "Human T-Lymphotropic Virus Type 1 p30II Regulates Gene Transcription by Binding CREB Binding Protein/p300"

    Article Title: Human T-Lymphotropic Virus Type 1 p30II Regulates Gene Transcription by Binding CREB Binding Protein/p300

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.20.9885-9895.2001

    p30 II inhibits the assembly of Tax-p300-CREB multiprotein complexes on TRE oligonucleotides. Exogenous Tax, p300, and p30 II -HA were introduced into 293T cells by transfection using expression plasmids. At 48 h posttransfection, nuclear lysates of transfected cells were incubated with the biotin-labeled TRE (HTLV-1 21-bp repeat) DNA probe as described in Materials and Methods. Streptavidin-agarose was used to isolate bound components. After extensive washing, bound proteins (right panel) were resolved using SDS-PAGE (4 to 20% gradient acrylamide) gels and detected by Western immunoblot assay. In parallel, equal amounts of nuclear lysates used as input were examined by Western immunoblot assay to determine protein expression levels of each component in transfected cells (left panel).
    Figure Legend Snippet: p30 II inhibits the assembly of Tax-p300-CREB multiprotein complexes on TRE oligonucleotides. Exogenous Tax, p300, and p30 II -HA were introduced into 293T cells by transfection using expression plasmids. At 48 h posttransfection, nuclear lysates of transfected cells were incubated with the biotin-labeled TRE (HTLV-1 21-bp repeat) DNA probe as described in Materials and Methods. Streptavidin-agarose was used to isolate bound components. After extensive washing, bound proteins (right panel) were resolved using SDS-PAGE (4 to 20% gradient acrylamide) gels and detected by Western immunoblot assay. In parallel, equal amounts of nuclear lysates used as input were examined by Western immunoblot assay to determine protein expression levels of each component in transfected cells (left panel).

    Techniques Used: Transfection, Expressing, Incubation, Labeling, SDS Page, Western Blot

    34) Product Images from "Focal Adhesions Require Catalytic Activity of Src Family Kinases To Mediate Integrin-Matrix Adhesion"

    Article Title: Focal Adhesions Require Catalytic Activity of Src Family Kinases To Mediate Integrin-Matrix Adhesion

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.4.1203-1217.2002

    The effect of FA-Csk on cell adhesion is independent of caspase-mediated apoptosis. (A) The effect of a membrane-anchored caspase domain of caspase 8 (CD8-C) on cell adhesion was determined with or without a caspase 3 inhibitor. After lipofection of NIH 3T3 cells (plated on fibronectin) with various amounts of pCEFL-CD8-C and 0.1 μg of a GFP expression plasmid, cells were cultured for 18 h with or without 8 μg of Z-DEVD-FMK (Calbiochem) per ml in DMEM containing 10% calf serum. (B) The effect of the caspase inhibitor Z-DEVD-FMK on cell detachment caused by FA-Csk(+) was determined. After lipofection of NIH 3T3 cells (plated on fibronectin) with kinase-active or inactive CskGFP-FAT, cells were cultured for 18 h in DMEM containing 10% calf serum and various doses of the caspase inhibitor. (C) Effect of activated (myristoylated) Akt on cell detachment caused by CD8-C expression. The number of GFP-positive cells remaining on coverslips in the group transfected with 0.3 μg of pCEFL-CD8-C is shown as a percentage of control (CTR) (that is, the mock-transfected group represents 100%) in parental NIH 3T3 cells as well as in those overexpressing a myristoylated mutant of Akt (NIH 3T3-myrAKT). All groups were cotransfected with a GFP expression vector. The Western blot above the bars shows the level of total Akt (endogenous and transfected) in NIH 3T3-myrAKT cells (right lane) compared to that of endogenous Akt in parental NIH 3T3 cells (left lane). (D) Effect of FA-Csk (0.1 μg of kinase-active or -inactive CskGFP-FAT plasmid) in NIH 3T3-myrAKT cells plated on fibronectin. (A to D) Values (means ± standard deviations from a triplicate experiment) indicate the numbers of transfected (GFP fluorescence-positive) cells remaining on the fibronectin-coated surface in 30 fields under a 25× objective (for panel C, values are given as percentages of control cells as noted above). (E) DNA fragmentation and chromatin condensation were determined for NIH 3T3 cells expressing FA-Csk(+) or FA-Csk(−) (kinase-active or -inactive CskGFP-FAT) plated on PLL/fibronectin double-coated coverslips 18 h after transfection. Each column shows one field of cells in three different channels (TUNEL, DAPI, and GFP signals). The arrows indicate the GFP-positive transfected cell in each group. Even after blocking endogenous biotinylated proteins, some background s taining can be seen in mitochondria in TUNEL staining. The nuclei were stained with DAPI. All experiments were repeated at least three times (A to E).
    Figure Legend Snippet: The effect of FA-Csk on cell adhesion is independent of caspase-mediated apoptosis. (A) The effect of a membrane-anchored caspase domain of caspase 8 (CD8-C) on cell adhesion was determined with or without a caspase 3 inhibitor. After lipofection of NIH 3T3 cells (plated on fibronectin) with various amounts of pCEFL-CD8-C and 0.1 μg of a GFP expression plasmid, cells were cultured for 18 h with or without 8 μg of Z-DEVD-FMK (Calbiochem) per ml in DMEM containing 10% calf serum. (B) The effect of the caspase inhibitor Z-DEVD-FMK on cell detachment caused by FA-Csk(+) was determined. After lipofection of NIH 3T3 cells (plated on fibronectin) with kinase-active or inactive CskGFP-FAT, cells were cultured for 18 h in DMEM containing 10% calf serum and various doses of the caspase inhibitor. (C) Effect of activated (myristoylated) Akt on cell detachment caused by CD8-C expression. The number of GFP-positive cells remaining on coverslips in the group transfected with 0.3 μg of pCEFL-CD8-C is shown as a percentage of control (CTR) (that is, the mock-transfected group represents 100%) in parental NIH 3T3 cells as well as in those overexpressing a myristoylated mutant of Akt (NIH 3T3-myrAKT). All groups were cotransfected with a GFP expression vector. The Western blot above the bars shows the level of total Akt (endogenous and transfected) in NIH 3T3-myrAKT cells (right lane) compared to that of endogenous Akt in parental NIH 3T3 cells (left lane). (D) Effect of FA-Csk (0.1 μg of kinase-active or -inactive CskGFP-FAT plasmid) in NIH 3T3-myrAKT cells plated on fibronectin. (A to D) Values (means ± standard deviations from a triplicate experiment) indicate the numbers of transfected (GFP fluorescence-positive) cells remaining on the fibronectin-coated surface in 30 fields under a 25× objective (for panel C, values are given as percentages of control cells as noted above). (E) DNA fragmentation and chromatin condensation were determined for NIH 3T3 cells expressing FA-Csk(+) or FA-Csk(−) (kinase-active or -inactive CskGFP-FAT) plated on PLL/fibronectin double-coated coverslips 18 h after transfection. Each column shows one field of cells in three different channels (TUNEL, DAPI, and GFP signals). The arrows indicate the GFP-positive transfected cell in each group. Even after blocking endogenous biotinylated proteins, some background s taining can be seen in mitochondria in TUNEL staining. The nuclei were stained with DAPI. All experiments were repeated at least three times (A to E).

    Techniques Used: Expressing, Plasmid Preparation, Cell Culture, Transfection, Mutagenesis, Western Blot, Fluorescence, TUNEL Assay, Blocking Assay, Staining

    35) Product Images from "NicE-seq: high resolution open chromatin profiling"

    Article Title: NicE-seq: high resolution open chromatin profiling

    Journal: Genome Biology

    doi: 10.1186/s13059-017-1247-6

    Nicking enzyme-mediated labeling of open chromatin. a Nicking of crosslinked chromatin using varying amounts of Nt.CviPII. A 1% agarose gel showing differential nicking of HCT116 genomic DNA based on amount of nicking enzyme (10 U, 5 U, 2.5 U, 1 U, 0.3 U, 0 U). M is a DNA molecular weight ladder. b Open chromatin labeling in fixed HeLa cells using dNTPs supplemented with TexasRed-dATP. Top panel : labeling reaction performed in the presence of Nt.CviPII and DNA polymerase I. Middle panel : labeling reaction performed in the presence of DNA polymerase I only. Bottom panel : labeling reaction performed in the absence of Nt.CviPII and DNA polymerase I. TexasRed-dATP was included in all reactions. DNA staining was performed using DAPI ( blue ) and TexasRed stain ( red ) represents labeled OCSs. Magenta stain ( Merge ) represents the colocalization. c Labeling efficiency of OCSs in all three assayed conditions. The y-axis represents the ratio of the intensity of the red pixels to the intensity of the blue pixels ( OCS labeling efficiency ). d Dot blot showing labeling of open chromatin by Nt.CviPII nicking enzyme in both native and formaldehyde-fixed HCT116 cells. The level of labeling was revealed using HRP-conjugated goat anti-biotin antibody
    Figure Legend Snippet: Nicking enzyme-mediated labeling of open chromatin. a Nicking of crosslinked chromatin using varying amounts of Nt.CviPII. A 1% agarose gel showing differential nicking of HCT116 genomic DNA based on amount of nicking enzyme (10 U, 5 U, 2.5 U, 1 U, 0.3 U, 0 U). M is a DNA molecular weight ladder. b Open chromatin labeling in fixed HeLa cells using dNTPs supplemented with TexasRed-dATP. Top panel : labeling reaction performed in the presence of Nt.CviPII and DNA polymerase I. Middle panel : labeling reaction performed in the presence of DNA polymerase I only. Bottom panel : labeling reaction performed in the absence of Nt.CviPII and DNA polymerase I. TexasRed-dATP was included in all reactions. DNA staining was performed using DAPI ( blue ) and TexasRed stain ( red ) represents labeled OCSs. Magenta stain ( Merge ) represents the colocalization. c Labeling efficiency of OCSs in all three assayed conditions. The y-axis represents the ratio of the intensity of the red pixels to the intensity of the blue pixels ( OCS labeling efficiency ). d Dot blot showing labeling of open chromatin by Nt.CviPII nicking enzyme in both native and formaldehyde-fixed HCT116 cells. The level of labeling was revealed using HRP-conjugated goat anti-biotin antibody

    Techniques Used: Labeling, Agarose Gel Electrophoresis, Molecular Weight, Staining, Dot Blot

    36) Product Images from "Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue"

    Article Title: Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2011.01.006

    Validating biotin-14-dCTP incorporation by a bind and boil method. The biotin–streptavidin dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.
    Figure Legend Snippet: Validating biotin-14-dCTP incorporation by a bind and boil method. The biotin–streptavidin dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.

    Techniques Used: Polymerase Chain Reaction, Amplification, Buffer Exchange, Chromatin Immunoprecipitation

    Pictorial representation of Washington University Capture. Washington University Capture (WUCap) enables solution-phase hybridization between double-stranded DNA PCR “bait” and whole-genome shotgun libraries. The solution-phase method we have developed for hybrid capture is robust and involves only a few basic steps. The bait used for targeting is dictated by primer-specific amplification of genomic targets generated during the PCR. Subsequently, the amplicons are used as a template in a second PCR incorporating biotin-14-dCTP. Genomic DNA is prepared from each of the samples to be sequenced, sheared to an average fragment size of 300 bp, enzymatically repaired to blunt the ends, and ligated to Illumina adapter sequences (at both ends). Five hundred nanograms of genomic DNA library is denatured, combined with 100 ng of the biotinylated “bait,” and hybridized for 48 hours. Mixing this hybridization reaction with streptavidin-coated superparamagnetic beads allows binding of biotinylated bait–target hybrids and selective removal from solution by applying a magnet field. The remaining supernatant is removed, and the beads are washed, removing nonspecific DNA. The enriched target sequences are released from the bead-bound bait sequences by denaturation (0.125 N NaOH), neutralized, amplified in the PCR to generate double-stranded Illumina libraries, and then sequenced.
    Figure Legend Snippet: Pictorial representation of Washington University Capture. Washington University Capture (WUCap) enables solution-phase hybridization between double-stranded DNA PCR “bait” and whole-genome shotgun libraries. The solution-phase method we have developed for hybrid capture is robust and involves only a few basic steps. The bait used for targeting is dictated by primer-specific amplification of genomic targets generated during the PCR. Subsequently, the amplicons are used as a template in a second PCR incorporating biotin-14-dCTP. Genomic DNA is prepared from each of the samples to be sequenced, sheared to an average fragment size of 300 bp, enzymatically repaired to blunt the ends, and ligated to Illumina adapter sequences (at both ends). Five hundred nanograms of genomic DNA library is denatured, combined with 100 ng of the biotinylated “bait,” and hybridized for 48 hours. Mixing this hybridization reaction with streptavidin-coated superparamagnetic beads allows binding of biotinylated bait–target hybrids and selective removal from solution by applying a magnet field. The remaining supernatant is removed, and the beads are washed, removing nonspecific DNA. The enriched target sequences are released from the bead-bound bait sequences by denaturation (0.125 N NaOH), neutralized, amplified in the PCR to generate double-stranded Illumina libraries, and then sequenced.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Amplification, Generated, Binding Assay

    37) Product Images from "Direct fluorescent labelling of clones by DOP PCR"

    Article Title: Direct fluorescent labelling of clones by DOP PCR

    Journal: Molecular Cytogenetics

    doi: 10.1186/1755-8166-1-3

    Results of FISH . A: DOP amplified BAC clone (BAC RP11-393B19) labelled with SpectrumOrange™- dUTP by means of the DOP4 primer in a PCR amplification reaction; B: DOP amplified BAC clone (BAC RP11-393B19) labelled with SpectrumOrange™- dUTP with the optimized protocol; C: DOP amplified BAC clone (BAC RP11-303I4) labelled with biotin d-CTP using the DOP4 primer in a PCR amplification reaction.; D: DOP amplified BAC clone (BAC RP11-303I4) labelled with biotin d-CTP with the optimized protocol.
    Figure Legend Snippet: Results of FISH . A: DOP amplified BAC clone (BAC RP11-393B19) labelled with SpectrumOrange™- dUTP by means of the DOP4 primer in a PCR amplification reaction; B: DOP amplified BAC clone (BAC RP11-393B19) labelled with SpectrumOrange™- dUTP with the optimized protocol; C: DOP amplified BAC clone (BAC RP11-303I4) labelled with biotin d-CTP using the DOP4 primer in a PCR amplification reaction.; D: DOP amplified BAC clone (BAC RP11-303I4) labelled with biotin d-CTP with the optimized protocol.

    Techniques Used: Fluorescence In Situ Hybridization, Amplification, BAC Assay, Polymerase Chain Reaction

    38) Product Images from "Linear Assembly of a Human Y Chromosome Centromere"

    Article Title: Linear Assembly of a Human Y Chromosome Centromere

    Journal: Nature biotechnology

    doi: 10.1038/nbt.4109

    BAC-based Longboard nanopore sequencing strategy on the MinION (a) Optimized strategy to cut each circular BAC once with transposase, resulting in a linear and complete DNA fragment of the BAC for nanopore sequencing. (b) Yield plots of BAC DNA (RP11-648J18) provide enrichment supporting BAC lengths. (c) High quality BAC consensus sequences were generated by multiple alignment of 60 full length 1D reads (shown as blue and yellow for both orientations) sampled at random with 10 iterations, followed by polishing steps (green) with the entire nanopore long read data and Illumina data. (d) Representation 20 of the polished RP11-718M18 BAC consensus sequence. Blue arrowheads indicate the position and orientation of HORs. Purple tiles in yellow background mark the position of the Illumina-validated variants. Additional purple highlight extending from select illumina-validated variants are used to identify single nucleotide sequence variants and mark the site of the DYZ3 repeat structural variants (6 kb) in tandem.
    Figure Legend Snippet: BAC-based Longboard nanopore sequencing strategy on the MinION (a) Optimized strategy to cut each circular BAC once with transposase, resulting in a linear and complete DNA fragment of the BAC for nanopore sequencing. (b) Yield plots of BAC DNA (RP11-648J18) provide enrichment supporting BAC lengths. (c) High quality BAC consensus sequences were generated by multiple alignment of 60 full length 1D reads (shown as blue and yellow for both orientations) sampled at random with 10 iterations, followed by polishing steps (green) with the entire nanopore long read data and Illumina data. (d) Representation 20 of the polished RP11-718M18 BAC consensus sequence. Blue arrowheads indicate the position and orientation of HORs. Purple tiles in yellow background mark the position of the Illumina-validated variants. Additional purple highlight extending from select illumina-validated variants are used to identify single nucleotide sequence variants and mark the site of the DYZ3 repeat structural variants (6 kb) in tandem.

    Techniques Used: BAC Assay, Nanopore Sequencing, Generated, Sequencing

    39) Product Images from "Karyotype diversity and chromosomal organization of repetitive DNA in Tityus obscurus (Scorpiones, Buthidae)"

    Article Title: Karyotype diversity and chromosomal organization of repetitive DNA in Tityus obscurus (Scorpiones, Buthidae)

    Journal: BMC Genetics

    doi: 10.1186/s12863-017-0494-6

    Heterochromatin and multigenic family mapping in 2n = 16 specimens. a Cot -1 DNA. The insert in ( a ) shows a meiotic cell in pachytene. b DAPI staining. c CMA 3 staining. d FISH H3 histone probe showing terminal hybridization. e U2 snRNA gene maping in a 2n = 16 specimen, a translocation bearer. f U2 snRNA gene in a 2n =16 specimen with eight bivalents. Barr = 10 μm
    Figure Legend Snippet: Heterochromatin and multigenic family mapping in 2n = 16 specimens. a Cot -1 DNA. The insert in ( a ) shows a meiotic cell in pachytene. b DAPI staining. c CMA 3 staining. d FISH H3 histone probe showing terminal hybridization. e U2 snRNA gene maping in a 2n = 16 specimen, a translocation bearer. f U2 snRNA gene in a 2n =16 specimen with eight bivalents. Barr = 10 μm

    Techniques Used: Staining, Fluorescence In Situ Hybridization, Hybridization, Translocation Assay

    40) Product Images from "Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray"

    Article Title: Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000924

    Strategy for amplification and labeling of pathogen-specific target. The one-tube RT-PCR reaction includes three stages: A) Reverse transcription reaction to produce first strand cDNA for viral targets (vRNA); B) Low temperature (56°C), exponential amplification of target and C) High temperature (68°C), linear amplification to product single-strand, biotin-labeled target. Target is labeled by incorporation of biotin-14-dCTP during amplification or by the use of biotinylated reverse primers.
    Figure Legend Snippet: Strategy for amplification and labeling of pathogen-specific target. The one-tube RT-PCR reaction includes three stages: A) Reverse transcription reaction to produce first strand cDNA for viral targets (vRNA); B) Low temperature (56°C), exponential amplification of target and C) High temperature (68°C), linear amplification to product single-strand, biotin-labeled target. Target is labeled by incorporation of biotin-14-dCTP during amplification or by the use of biotinylated reverse primers.

    Techniques Used: Amplification, Labeling, Reverse Transcription Polymerase Chain Reaction

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    Article Snippet: .. Plasmid DNA was labelled by nick translation using biotin-14-dATP of the Bionick™ Labeling System (Invitrogen Life Technologies, Paisley, UK). .. Plasmid DNA (0.5–1 μg) was labelled using 50 μL of labelling reaction (using 5 μL of 10× enzyme mix) for 2 h at 16 °C, and the reaction was stopped with 5 μL of 500 mM EDTA (pH 8.0).

    Article Title: Presence and integration of HBV DNA in mouse oocytes
    Article Snippet: .. The 3.2 kb HBV DNA probe was labeled with biotin-14-dATP by nick translation (BioNick DNA Labeling System, GIBCO BRL). .. Unincorporated nucleotides were separated by cold ethanol precipitation method.

    Article Title: First record of supernumerary (B) chromosomes in electric fish (Gymnotiformes) and the karyotype structure of three species of the same order from the upper Paran? River basin
    Article Snippet: .. The probes were labelled with biotin 14-dATP via nick translation (Bio Nick Labeling System - Gibco, BRL). .. We incubated the slides with RNase (37 ºC, 1 h) and then treated them with 30 μl of hybridization mixture containing 100–300 ng of labelled probes (3 μl), 15 μl 100% formamide, 20×SSC (0.5 μl), 0.1 μl calf thymus DNA, 0.6 μl 50% Dextran and 0.1 μl 10% SDS.

    Article Title: Description of karyotype in Hypostomus regani (Ihering, 1905) (Teleostei, oricariidae) from the Piumhi river in Brazil with comments on karyotype variation found in Hypostomus
    Article Snippet: .. Both probes were labeled with 14-dATP biotin by nick translation, following the manufacturer’s instructions (Bionick Labeling System – Invitrogen). .. Amplification and detection of the hybridization signals was carried out using the avidin-FITC and anti-avidin biotin complex system (Sigma).

    Article Title: Uncovering the evolutionary history of neo-XY sex chromosomes in the grasshopper Ronderosia bergii (Orthoptera, Melanoplinae) through satellite DNA analysis
    Article Snippet: .. Probes and Fluorescence In situ Hybridization, measurement of sex chromosomes and distance between signals The PCR products for each satDNA family with more than 50 bp were labeled by nick translation using biotin-14-dATP (Invitrogen) or digoxigenin-11-dUTP (Roche, Mannheim, Germany). .. SatDNAs with less than 50 bp were labeled directly at the 5′ end with biotin-14 dATP (Sigma-Aldrich, St Louis, MO, USA) during their synthesis.

    Article Title: CDK inhibitor p21 is prosurvival in adriamycin-induced podocyte injury, in vitro and in vivo
    Article Snippet: .. Following incubation in One-Phor-All buffer (GE Healthcare, Piscataway, NJ), fragmented DNA was labeled by exposure of sections to diluted TdT (GE Healthcare) and biotin-14-dATP (Invitrogen, Grand Island, NY) for 60 min. ..

    Incubation:

    Article Title: CDK inhibitor p21 is prosurvival in adriamycin-induced podocyte injury, in vitro and in vivo
    Article Snippet: .. Following incubation in One-Phor-All buffer (GE Healthcare, Piscataway, NJ), fragmented DNA was labeled by exposure of sections to diluted TdT (GE Healthcare) and biotin-14-dATP (Invitrogen, Grand Island, NY) for 60 min. ..

    DNA Labeling:

    Article Title: Presence and integration of HBV DNA in mouse oocytes
    Article Snippet: .. The 3.2 kb HBV DNA probe was labeled with biotin-14-dATP by nick translation (BioNick DNA Labeling System, GIBCO BRL). .. Unincorporated nucleotides were separated by cold ethanol precipitation method.

    Polymerase Chain Reaction:

    Article Title: Uncovering the evolutionary history of neo-XY sex chromosomes in the grasshopper Ronderosia bergii (Orthoptera, Melanoplinae) through satellite DNA analysis
    Article Snippet: .. Probes and Fluorescence In situ Hybridization, measurement of sex chromosomes and distance between signals The PCR products for each satDNA family with more than 50 bp were labeled by nick translation using biotin-14-dATP (Invitrogen) or digoxigenin-11-dUTP (Roche, Mannheim, Germany). .. SatDNAs with less than 50 bp were labeled directly at the 5′ end with biotin-14 dATP (Sigma-Aldrich, St Louis, MO, USA) during their synthesis.

    In Situ Hybridization:

    Article Title: Uncovering the evolutionary history of neo-XY sex chromosomes in the grasshopper Ronderosia bergii (Orthoptera, Melanoplinae) through satellite DNA analysis
    Article Snippet: .. Probes and Fluorescence In situ Hybridization, measurement of sex chromosomes and distance between signals The PCR products for each satDNA family with more than 50 bp were labeled by nick translation using biotin-14-dATP (Invitrogen) or digoxigenin-11-dUTP (Roche, Mannheim, Germany). .. SatDNAs with less than 50 bp were labeled directly at the 5′ end with biotin-14 dATP (Sigma-Aldrich, St Louis, MO, USA) during their synthesis.

    Plasmid Preparation:

    Article Title: Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don
    Article Snippet: .. Plasmid DNA was labelled by nick translation using biotin-14-dATP of the Bionick™ Labeling System (Invitrogen Life Technologies, Paisley, UK). .. Plasmid DNA (0.5–1 μg) was labelled using 50 μL of labelling reaction (using 5 μL of 10× enzyme mix) for 2 h at 16 °C, and the reaction was stopped with 5 μL of 500 mM EDTA (pH 8.0).

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    Thermo Fisher biotin 14 datp
    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, <t>biotin-14-dCTP,</t> and <t>biotin-14-dATP,</t> are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.
    Biotin 14 Datp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotin 14 datp/product/Thermo Fisher
    Average 98 stars, based on 83 article reviews
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    biotin 14 datp - by Bioz Stars, 2020-08
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    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    doi: 10.3389/fbioe.2019.00401

    Figure Lengend Snippet: Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Article Snippet: Biotin-14-dCTP and biotin-14-dATP were obtained from Thermo Scientific Co., Ltd. (Shanghai, China).

    Techniques: Labeling, Amplification

    Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    doi: 10.3389/fbioe.2019.00401

    Figure Lengend Snippet: Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Article Snippet: Biotin-14-dCTP and biotin-14-dATP were obtained from Thermo Scientific Co., Ltd. (Shanghai, China).

    Techniques: