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  • 99
    Millipore dna amplification
    SNP analysis of centromeric domains. Sanger sequence traces from input ( above ) and CENP-A <t>immunoprecipitated</t> ( below ) samples from HSF-C, HSF-G, HSF-D and HSF-E. SNP coordinates are beneath traces. Stars indicate SNPs. For HSF-C, HSF-G, HSF-D-edge and HSF-E-edge, both nucleotides are present in input <t>DNA</t> while the immunoprecipitated DNA is enriched for one of the two nucleotides. For HSF-D centre and HSF-E centre, the two nucleotides are present in both input and CENP-A immunoprecipitated samples
    Dna Amplification, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna amplifications
    Suggested <t>DNA/RNA</t> co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Dna Amplifications, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 698 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna amplifications/product/Millipore
    Average 99 stars, based on 698 article reviews
    Price from $9.99 to $1999.99
    dna amplifications - by Bioz Stars, 2020-08
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    99
    Bio-Rad deoxyribonucleic acid dna amplification
    Suggested <t>DNA/RNA</t> co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Deoxyribonucleic Acid Dna Amplification, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore whole genome amplified dna
    Suggested <t>DNA/RNA</t> co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Whole Genome Amplified Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SNP analysis of centromeric domains. Sanger sequence traces from input ( above ) and CENP-A immunoprecipitated ( below ) samples from HSF-C, HSF-G, HSF-D and HSF-E. SNP coordinates are beneath traces. Stars indicate SNPs. For HSF-C, HSF-G, HSF-D-edge and HSF-E-edge, both nucleotides are present in input DNA while the immunoprecipitated DNA is enriched for one of the two nucleotides. For HSF-D centre and HSF-E centre, the two nucleotides are present in both input and CENP-A immunoprecipitated samples

    Journal: Chromosoma

    Article Title: Centromere sliding on a mammalian chromosome

    doi: 10.1007/s00412-014-0493-6

    Figure Lengend Snippet: SNP analysis of centromeric domains. Sanger sequence traces from input ( above ) and CENP-A immunoprecipitated ( below ) samples from HSF-C, HSF-G, HSF-D and HSF-E. SNP coordinates are beneath traces. Stars indicate SNPs. For HSF-C, HSF-G, HSF-D-edge and HSF-E-edge, both nucleotides are present in input DNA while the immunoprecipitated DNA is enriched for one of the two nucleotides. For HSF-D centre and HSF-E centre, the two nucleotides are present in both input and CENP-A immunoprecipitated samples

    Article Snippet: Both input and immunoprecipitated DNA fragments were purified and amplified using the whole genome amplification (WGA) kit (Sigma-Aldrich, St. Louis, USA).

    Techniques: Sequencing, Immunoprecipitation

    Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x -axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3 ). b Peak positions are represented as boxes . Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3 ) and fibre FISH (Fig. 4 and Supplementary Table 2 ) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser ( http://genome.ucsc.edu ). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4 ) and SNP analysis (Fig. 3 and Supplementary Table 2 ), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb

    Journal: Chromosoma

    Article Title: Centromere sliding on a mammalian chromosome

    doi: 10.1007/s00412-014-0493-6

    Figure Lengend Snippet: Variable position of the centromere of horse chromosome 11. a DNA obtained by chromatin immunoprecipitation. Using an anti-CENP-A antibody, from five different horse fibroblast cultures was hybridized to a tiling array covering the centromere region. Results are presented as the log2 ratio of the hybridization signals obtained with immunoprecipitated DNA versus input DNA; x -axis, genomic coordinates on ECA11. Positions of informative SNPs are indicated as black dots (a single nucleotide of the SNP is enriched in immunoprecipitated DNA), red dots (both SNP alleles are present in immunoprecipitated DNA) and blue carats (SNPs shown in Fig. 3 ). b Peak positions are represented as boxes . Epiallele identification was obtained by combining ChIP-on-chip, SNP (Fig. 3 ) and fibre FISH (Fig. 4 and Supplementary Table 2 ) results. Sequence coordinates refer to the horse EquCab2.0 (2007) sequence assembly, as reported by the UCSC genome browser ( http://genome.ucsc.edu ). Alleles are designated by the letter of the horse they derive from, followed by ‘1’ or ‘2’ to distinguish the two variants. In HSF-D and HSF-E, where a single broad peak was identified by ChIP-on-chip while two distinct centromeric domains were identified by fibre-FISH (Fig. 4 ) and SNP analysis (Fig. 3 and Supplementary Table 2 ), dotted lines represent the region of overlap of the two binding domains in the reference sequence. Therefore, at least seven different centromeric domains can be identified: Ba/Ea, Bb, Ca, Cb, Da/Eb, Db/Ga, Gb

    Article Snippet: Both input and immunoprecipitated DNA fragments were purified and amplified using the whole genome amplification (WGA) kit (Sigma-Aldrich, St. Louis, USA).

    Techniques: Chromatin Immunoprecipitation, Hybridization, Immunoprecipitation, Fluorescence In Situ Hybridization, Sequencing, Binding Assay

    Procedure of T oligo-primed polymerase chain reaction (TOP-PCR). Half adaptor is generated by annealing P and T oligos. P oligo carries a 5′ phosphate required for ligation to target DNA fragments, while T oligo does not. During amplification, only the T oligo serves as the PCR primer. Number of amplification cycle depends on the initial concentration of the target DNA molecules and the desired final quantity.

    Journal: Scientific Reports

    Article Title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids

    doi: 10.1038/srep40767

    Figure Lengend Snippet: Procedure of T oligo-primed polymerase chain reaction (TOP-PCR). Half adaptor is generated by annealing P and T oligos. P oligo carries a 5′ phosphate required for ligation to target DNA fragments, while T oligo does not. During amplification, only the T oligo serves as the PCR primer. Number of amplification cycle depends on the initial concentration of the target DNA molecules and the desired final quantity.

    Article Snippet: The reaction mixture (50 μL) contains 10 μL DNA template (in desired quantity), 0.64 μM T oligo, 1.5 mM MgSO4 , 0.2 mM dNTPs, 1 X buffer, and 1unit Novagen KOD hot start DNA polymerase (EMD Millipore Co., 71086-3).

    Techniques: Polymerase Chain Reaction, Generated, Ligation, Amplification, Concentration Assay

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    Schematic diagram of the optimization process. In Stage 1 of the process, various extraction kits and Nicolaisen’s method (as listed in Table 1 ) was tested on soils FH and FL (see text for soil descriptions). In Stage 2, various extraction buffers, lysis conditions, and nucleic acid precipitants were tested using Nicolaisen’s method as the base, creating a new “semi-optimized Nicolaisen’s method.” In the final Stage 3, DNases/reverse transcriptases and purification kits were tested concurrently for their ability to completely remove genomic DNA, and was briefly tested in combination. The end result is the “Modified Nicolaisen’s method,” which is based on the workflow as outlined in Figure 2 .

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Schematic diagram of the optimization process. In Stage 1 of the process, various extraction kits and Nicolaisen’s method (as listed in Table 1 ) was tested on soils FH and FL (see text for soil descriptions). In Stage 2, various extraction buffers, lysis conditions, and nucleic acid precipitants were tested using Nicolaisen’s method as the base, creating a new “semi-optimized Nicolaisen’s method.” In the final Stage 3, DNases/reverse transcriptases and purification kits were tested concurrently for their ability to completely remove genomic DNA, and was briefly tested in combination. The end result is the “Modified Nicolaisen’s method,” which is based on the workflow as outlined in Figure 2 .

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Lysis, Purification

    Removal of gDNA by consecutive DNase digestions of total nucleic acids (TNA) extracted from 45 Å soil samples. The soil had been exposed to different oxygen regimes (here called Treatments 1, 2, and 3), for details see section “Materials and Methods.” The soils were incubated anoxically to stimulate denitrification gene expression, and samples were taken at time int ervals. TNA was extracted using the optimized and simplified method, and the nosZ was quantified by qPCR. (A) After extraction via the optimized method, all samples were tested for the presence of DNA. Neither the different oxygen regimes nor the stimulation of gene expression affected the number of nosZ genes in the gDNA from the different samples. (B) The first digest removed most amplifiable genomic DNA (gDNA) present. (C) The second DNase treatment removed amplifiable gDNA in all samples. There was no relationship between the starting DNA quantity and the success of complete gDNA removal ( R 2 = 0.0189). This highlights the importance of checking all RNA samples and not only representative samples, as there may be high variability among samples from the same source and extraction procedure.

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Removal of gDNA by consecutive DNase digestions of total nucleic acids (TNA) extracted from 45 Å soil samples. The soil had been exposed to different oxygen regimes (here called Treatments 1, 2, and 3), for details see section “Materials and Methods.” The soils were incubated anoxically to stimulate denitrification gene expression, and samples were taken at time int ervals. TNA was extracted using the optimized and simplified method, and the nosZ was quantified by qPCR. (A) After extraction via the optimized method, all samples were tested for the presence of DNA. Neither the different oxygen regimes nor the stimulation of gene expression affected the number of nosZ genes in the gDNA from the different samples. (B) The first digest removed most amplifiable genomic DNA (gDNA) present. (C) The second DNase treatment removed amplifiable gDNA in all samples. There was no relationship between the starting DNA quantity and the success of complete gDNA removal ( R 2 = 0.0189). This highlights the importance of checking all RNA samples and not only representative samples, as there may be high variability among samples from the same source and extraction procedure.

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Incubation, Expressing, Real-time Polymerase Chain Reaction

    Loop mediated isothermal amplification of T. foetus DNA. (A) Timecourse of LAMP reaction: lane M, 50-bp ladder; the following lanes show LAMP products at 0, 30, 60, 90 and 120 min. (B) Visual inspection: test tubes under UV light containing the same amplification products as in (A). Positive reactions turned bright green upon addition of SYBR Green I, while the negative ones remained dark under UV light. (C) Digestion of T. foetus LAMP amplification products with PauI (BssHII) developed in 2% agarose gel (left) and silver stained 10% non-denaturing PAGE (right). M, 50-bp ladder; (+) restriction products (1 μl); (−) non digested product (5 μl). Arrowheads point to digestion products with expected sizes of 113 and 135 base pairs.

    Journal: Veterinary Parasitology

    Article Title: Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus

    doi: 10.1016/j.vetpar.2012.11.034

    Figure Lengend Snippet: Loop mediated isothermal amplification of T. foetus DNA. (A) Timecourse of LAMP reaction: lane M, 50-bp ladder; the following lanes show LAMP products at 0, 30, 60, 90 and 120 min. (B) Visual inspection: test tubes under UV light containing the same amplification products as in (A). Positive reactions turned bright green upon addition of SYBR Green I, while the negative ones remained dark under UV light. (C) Digestion of T. foetus LAMP amplification products with PauI (BssHII) developed in 2% agarose gel (left) and silver stained 10% non-denaturing PAGE (right). M, 50-bp ladder; (+) restriction products (1 μl); (−) non digested product (5 μl). Arrowheads point to digestion products with expected sizes of 113 and 135 base pairs.

    Article Snippet: 2.7 Detection of amplified LAMP products LAMP DNA amplification was assessed through agarose gel electrophoresis or by direct visualization after addition of SYBR Green I (Sigma–Aldrich).

    Techniques: Amplification, SYBR Green Assay, Agarose Gel Electrophoresis, Staining, Polyacrylamide Gel Electrophoresis

    Specificity of T. foetus LAMP. (A) DNA from Trichomonads strains were amplified through PCR (primers TFR1 and TFR2 for genus recognition) and developed on 5% non-denaturing PAGE. Lane M, 50-bp ladder used as a size marker; lanes 1, 2, 3 and 4 Tetratrichomonas spp . strains GM018, GM019, GM020 and GM022 respectively; lanes 5, 6, 12, 13 and 14 T. foetus strains GM031, GM032, GM050, GM051 and GM052 respectively; lanes 7–10 Pentatrichomonas hominis strains GM033, GM034, GM035 and GM036 respectively; lane 11 mixed culture of Tetratrichomonas spp . GM022 and Pentatrichomonas hominis GM035. (B) The same samples as in (A) but with primers TFR3 and TFR4 (specific for T. foetus ). (C) Agarose gel analysis of LAMP specific for T. foetus 5.8S ribosomal gene. Lanes follow the same order as in (A) and (B). (D) LAMP reactions as in (C) after addition of SYBR green I.

    Journal: Veterinary Parasitology

    Article Title: Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus

    doi: 10.1016/j.vetpar.2012.11.034

    Figure Lengend Snippet: Specificity of T. foetus LAMP. (A) DNA from Trichomonads strains were amplified through PCR (primers TFR1 and TFR2 for genus recognition) and developed on 5% non-denaturing PAGE. Lane M, 50-bp ladder used as a size marker; lanes 1, 2, 3 and 4 Tetratrichomonas spp . strains GM018, GM019, GM020 and GM022 respectively; lanes 5, 6, 12, 13 and 14 T. foetus strains GM031, GM032, GM050, GM051 and GM052 respectively; lanes 7–10 Pentatrichomonas hominis strains GM033, GM034, GM035 and GM036 respectively; lane 11 mixed culture of Tetratrichomonas spp . GM022 and Pentatrichomonas hominis GM035. (B) The same samples as in (A) but with primers TFR3 and TFR4 (specific for T. foetus ). (C) Agarose gel analysis of LAMP specific for T. foetus 5.8S ribosomal gene. Lanes follow the same order as in (A) and (B). (D) LAMP reactions as in (C) after addition of SYBR green I.

    Article Snippet: 2.7 Detection of amplified LAMP products LAMP DNA amplification was assessed through agarose gel electrophoresis or by direct visualization after addition of SYBR Green I (Sigma–Aldrich).

    Techniques: Amplification, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Agarose Gel Electrophoresis, SYBR Green Assay

    Analytical sensitivity of T. foetus 5.8S rDNA LAMP. (A) DNA from T. foetus GM032 at different concentrations used for both LAMP (top) and PCR (bottom) assays. Detection limit ≈10 pg. (B) Sensitivity of LAMP assayed with crude DNA consisting of lysates of T. foetus cells in saline solution. Detection limit ≈4 × 10 3 cells/mL. (C) Sensitivity of LAMP assay for smegma spiked with different concentrations of T. foetus GM032. Detection limit ≈4 × 10 3 cells/mL. DNA (ng) and organism concentration (cells/mL) used for LAMP and PCR are indicated in the top of the gels. NC: negative control without DNA or without cells. The position of the molecular markers (from a 50 bp marker) are indicated in the left side of the gels. The results corresponded to three independent experiments were performed by triplicate.

    Journal: Veterinary Parasitology

    Article Title: Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus

    doi: 10.1016/j.vetpar.2012.11.034

    Figure Lengend Snippet: Analytical sensitivity of T. foetus 5.8S rDNA LAMP. (A) DNA from T. foetus GM032 at different concentrations used for both LAMP (top) and PCR (bottom) assays. Detection limit ≈10 pg. (B) Sensitivity of LAMP assayed with crude DNA consisting of lysates of T. foetus cells in saline solution. Detection limit ≈4 × 10 3 cells/mL. (C) Sensitivity of LAMP assay for smegma spiked with different concentrations of T. foetus GM032. Detection limit ≈4 × 10 3 cells/mL. DNA (ng) and organism concentration (cells/mL) used for LAMP and PCR are indicated in the top of the gels. NC: negative control without DNA or without cells. The position of the molecular markers (from a 50 bp marker) are indicated in the left side of the gels. The results corresponded to three independent experiments were performed by triplicate.

    Article Snippet: 2.7 Detection of amplified LAMP products LAMP DNA amplification was assessed through agarose gel electrophoresis or by direct visualization after addition of SYBR Green I (Sigma–Aldrich).

    Techniques: Polymerase Chain Reaction, Lamp Assay, Concentration Assay, Negative Control, Marker

    CN profiles of unamplified (UA) and WGA samples. a CN profile of MCC sample MCT4, which is unamplified and untreated, with 100 ng input of DNA. b CN profile of the same sample, which is unamplified and untreated, down-sampled to the similar coverage as the matched WGA sample. c CN profile of the same sample, which is WGA and untreated. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]

    Journal: Genome Medicine

    Article Title: Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

    doi: 10.1186/s13073-016-0375-z

    Figure Lengend Snippet: CN profiles of unamplified (UA) and WGA samples. a CN profile of MCC sample MCT4, which is unamplified and untreated, with 100 ng input of DNA. b CN profile of the same sample, which is unamplified and untreated, down-sampled to the similar coverage as the matched WGA sample. c CN profile of the same sample, which is WGA and untreated. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]

    Article Snippet: Whole genome amplification Extracted DNA from FFPE MCC samples were amplified using GenomePlex® Complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich), following the manufacturer’s instruction with several minor modifications.

    Techniques: Whole Genome Amplification

    Experimental design testing a low-input method (NEBNext Ultra II) on copy number detection of two FFPE MCC samples by LC WGS as well as the effect of WGA with or without NEB DNA repair treatment

    Journal: Genome Medicine

    Article Title: Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

    doi: 10.1186/s13073-016-0375-z

    Figure Lengend Snippet: Experimental design testing a low-input method (NEBNext Ultra II) on copy number detection of two FFPE MCC samples by LC WGS as well as the effect of WGA with or without NEB DNA repair treatment

    Article Snippet: Whole genome amplification Extracted DNA from FFPE MCC samples were amplified using GenomePlex® Complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich), following the manufacturer’s instruction with several minor modifications.

    Techniques: Formalin-fixed Paraffin-Embedded, Whole Genome Amplification

    Copy number profiles of MCC sample MCT4 with DNA input of ( a ) 100 ng, ( b ) 20 ng, and ( c ) 5 ng. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]

    Journal: Genome Medicine

    Article Title: Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue

    doi: 10.1186/s13073-016-0375-z

    Figure Lengend Snippet: Copy number profiles of MCC sample MCT4 with DNA input of ( a ) 100 ng, ( b ) 20 ng, and ( c ) 5 ng. Each point represents the normalized read count ratio of a 50 kb sized bin. Separate chromosomes from 1 to 22 as well as X and Y are shown and a log 2 (copy number/2) equal to zero corresponds to a copy number of 2. Segments were removed from highly repetitive or problematic regions [ 8 ]

    Article Snippet: Whole genome amplification Extracted DNA from FFPE MCC samples were amplified using GenomePlex® Complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich), following the manufacturer’s instruction with several minor modifications.

    Techniques:

    PCR and Southern blot experiments showed that group II intron-based vectors efficiently targeted the Clostridium cellulolyticum mdh and ldh genes in pure cultures . (A) Primers MdhF/intronR1 (5' junction) and intronF1/MdhR (3' junction) produced bands from the Ccel_0137 mutant cells (lanes 1 and 2) but not from wild-type (lanes 4 and 5). Primers MdhF/MdhR amplified a single band from the mutant (lane 3) that is 915 bp larger than the wild-type (lane 6). (B) Primers LdhF/intronR1 (5' junction) and intronF1/LdhR (3' junction) produced bands in the Ccel_2485 mutant cells (lanes 1 and 2) but not in the wild-type (lanes 4 and 5). Primers LdhF/LdhR, amplified a single band from the mutant (lane 3), which is 915 bp larger than the wild-type (lane 6). (C) Amplifications using plasmid-specific primers pWH199F2 and pintronR1 confirmed plasmid curing. Lane 1, positive control (plasmid); lane 2, Ccel_0137 mutant; lane 3, Ccel_2485 mutant; lane 4, negative control. (D) Amplification from wild-type DNA using primers LdhF-R (lane 1) and MdhF-R (lane 2) produced low molecular weight products. Genes containing insertions were amplified from the ldh mutant using primers LdhF-R (lane 3) and from the mdh mutant using primers MdhF-R (lane 4), producing larger products. The same size PCR products were obtained in amplifications from ldh mdh mutant DNA using primers LdhF-R (lane 5) and MdhF-R (lane 6). (E) A Southern blot using an intron-specific probe confirmed the intron insertions in DNA digested with Eco RI. No band was detected in the chromosomal DNA of wild-type cells (lane 1), while two bands in the ldh mdh mutant (lane 2) correspond to bands in the ldh mutant (lane 3) and the mdh mutant (lane 4). No band corresponding to the plasmid (lane 5) was identified in any of the plasmid-cured strains.

    Journal: Biotechnology for Biofuels

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    doi: 10.1186/1754-6834-5-2

    Figure Lengend Snippet: PCR and Southern blot experiments showed that group II intron-based vectors efficiently targeted the Clostridium cellulolyticum mdh and ldh genes in pure cultures . (A) Primers MdhF/intronR1 (5' junction) and intronF1/MdhR (3' junction) produced bands from the Ccel_0137 mutant cells (lanes 1 and 2) but not from wild-type (lanes 4 and 5). Primers MdhF/MdhR amplified a single band from the mutant (lane 3) that is 915 bp larger than the wild-type (lane 6). (B) Primers LdhF/intronR1 (5' junction) and intronF1/LdhR (3' junction) produced bands in the Ccel_2485 mutant cells (lanes 1 and 2) but not in the wild-type (lanes 4 and 5). Primers LdhF/LdhR, amplified a single band from the mutant (lane 3), which is 915 bp larger than the wild-type (lane 6). (C) Amplifications using plasmid-specific primers pWH199F2 and pintronR1 confirmed plasmid curing. Lane 1, positive control (plasmid); lane 2, Ccel_0137 mutant; lane 3, Ccel_2485 mutant; lane 4, negative control. (D) Amplification from wild-type DNA using primers LdhF-R (lane 1) and MdhF-R (lane 2) produced low molecular weight products. Genes containing insertions were amplified from the ldh mutant using primers LdhF-R (lane 3) and from the mdh mutant using primers MdhF-R (lane 4), producing larger products. The same size PCR products were obtained in amplifications from ldh mdh mutant DNA using primers LdhF-R (lane 5) and MdhF-R (lane 6). (E) A Southern blot using an intron-specific probe confirmed the intron insertions in DNA digested with Eco RI. No band was detected in the chromosomal DNA of wild-type cells (lane 1), while two bands in the ldh mdh mutant (lane 2) correspond to bands in the ldh mutant (lane 3) and the mdh mutant (lane 4). No band corresponding to the plasmid (lane 5) was identified in any of the plasmid-cured strains.

    Article Snippet: To insert the intron into pWH199 downstream of a Clostridium pasteurianum ferredoxin (Fd) promoter [ ], a DNA fragment containing the intron and ltrA was amplified from pJIR750ai (Sigma-Aldrich, St. Louis, MO) by PCR using primers pJIR750aiXmaIF and pJIR750aiXhoIR (Additional file ).

    Techniques: Polymerase Chain Reaction, Southern Blot, Produced, Mutagenesis, Amplification, Plasmid Preparation, Positive Control, Negative Control, Molecular Weight

    (A) Northern analysis of the sarZ and mgrA transcripts in the wild type (Wt), various isogenic mutants, and a single-copy complemented strain of the mgrA mutant at exponential phase (OD 600 , ∼0.7) of growth. DNA fragments (500 bp and 550 bp) containing

    Journal:

    Article Title: sarZ, a sarA Family Gene, Is Transcriptionally Activated by MgrA and Is Involved in the Regulation of Genes Encoding Exoproteins in Staphylococcus aureus ▿

    doi: 10.1128/JB.01555-08

    Figure Lengend Snippet: (A) Northern analysis of the sarZ and mgrA transcripts in the wild type (Wt), various isogenic mutants, and a single-copy complemented strain of the mgrA mutant at exponential phase (OD 600 , ∼0.7) of growth. DNA fragments (500 bp and 550 bp) containing

    Article Snippet: The 450-bp DNA fragment containing the full-length sarZ gene was amplified by PCR using chromosomal DNA from S. aureus RN6390 as the template and primers containing flanking NdeI and BamHI restriction sites (5′-ATCATATGTATGTAGAAAACAGCTAT-3′ and 5′-ATGGATCCATACTTCTGCCCATCACCTTAT-3′) to facilitate in-frame cloning into the expression vector pET14b (Novagen, Madison, WI).

    Techniques: Northern Blot, Mutagenesis

    DNA binding activity of SarZ protein. Autoradiograms of 8.0% polyacrylamide gels showing the binding of SarZ protein to 287-bp and 250-bp promoter fragments (1 ng each or 5 and 6 fM, respectively) of sarS (A) and sspA (B), respectively. In panel

    Journal:

    Article Title: sarZ, a sarA Family Gene, Is Transcriptionally Activated by MgrA and Is Involved in the Regulation of Genes Encoding Exoproteins in Staphylococcus aureus ▿

    doi: 10.1128/JB.01555-08

    Figure Lengend Snippet: DNA binding activity of SarZ protein. Autoradiograms of 8.0% polyacrylamide gels showing the binding of SarZ protein to 287-bp and 250-bp promoter fragments (1 ng each or 5 and 6 fM, respectively) of sarS (A) and sspA (B), respectively. In panel

    Article Snippet: The 450-bp DNA fragment containing the full-length sarZ gene was amplified by PCR using chromosomal DNA from S. aureus RN6390 as the template and primers containing flanking NdeI and BamHI restriction sites (5′-ATCATATGTATGTAGAAAACAGCTAT-3′ and 5′-ATGGATCCATACTTCTGCCCATCACCTTAT-3′) to facilitate in-frame cloning into the expression vector pET14b (Novagen, Madison, WI).

    Techniques: Binding Assay, Activity Assay

    ( A ): The polymerase chain reaction results of hph gene (1200 bp) amplification; the inserted hph gene was detected with primers hph -F and hph -R. Lane 1: pDHt/ZafA::hph , Lane 2: ZafA-hph, Lane 3: ZafA+bar, Lane 4: The wild-type T. mentagrophytes strain. ( B ): The polymerase chain reaction results of bar (900 bp) gene amplification, the inserted bar gene was detected with primers bar- F and bar -R. Lane 1: pDHt/ZafA-bar , Lane 2: ZafA+bar, Lane 3: pDHt/ZafA::hph , Lane 4: ZafA-hph, Lane 5: The wild-type T. mentagrophytes strain. ( C ): The polymerase chain reaction results of ZafA gene (400 bp) amplification, the inserted ZafA gene was detected with primers ZafA q-F and ZafA q-R. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA-bar , Lane 3: ZafA+bar, Lane 4: pDHt/ZafA::hph , Lane 5: ZafA-hph. ( D ): The Southern blot results of ZafA gene, in which a partial ZafA gene fragment (304 bp) was used as a hybridization probe for detecting the deleted gene fragment. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA::hph , Lane 3: ZafA-hph, Lane M: DNA Molecular-Weight Marker, Lane 4: The wild-type T. mentagrophytes strain, Lane 5: pDHt/ZafA-bar , Lane 6: ZafA+bar, Lane 7: pDHt/ZafA::hph , Lane 8: ZafA-hph, Lane C: Positive fragments.

    Journal: International Journal of Molecular Sciences

    Article Title: ZafA Gene Is Important for Trichophyton mentagrophytes Growth and Pathogenicity

    doi: 10.3390/ijms20040848

    Figure Lengend Snippet: ( A ): The polymerase chain reaction results of hph gene (1200 bp) amplification; the inserted hph gene was detected with primers hph -F and hph -R. Lane 1: pDHt/ZafA::hph , Lane 2: ZafA-hph, Lane 3: ZafA+bar, Lane 4: The wild-type T. mentagrophytes strain. ( B ): The polymerase chain reaction results of bar (900 bp) gene amplification, the inserted bar gene was detected with primers bar- F and bar -R. Lane 1: pDHt/ZafA-bar , Lane 2: ZafA+bar, Lane 3: pDHt/ZafA::hph , Lane 4: ZafA-hph, Lane 5: The wild-type T. mentagrophytes strain. ( C ): The polymerase chain reaction results of ZafA gene (400 bp) amplification, the inserted ZafA gene was detected with primers ZafA q-F and ZafA q-R. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA-bar , Lane 3: ZafA+bar, Lane 4: pDHt/ZafA::hph , Lane 5: ZafA-hph. ( D ): The Southern blot results of ZafA gene, in which a partial ZafA gene fragment (304 bp) was used as a hybridization probe for detecting the deleted gene fragment. Lane 1: The wild-type T. mentagrophytes strain, Lane 2: pDHt/ZafA::hph , Lane 3: ZafA-hph, Lane M: DNA Molecular-Weight Marker, Lane 4: The wild-type T. mentagrophytes strain, Lane 5: pDHt/ZafA-bar , Lane 6: ZafA+bar, Lane 7: pDHt/ZafA::hph , Lane 8: ZafA-hph, Lane C: Positive fragments.

    Article Snippet: Total DNA from the wild-type T. mentagrophytes strain, ZafA+hph, and ZafA+bar were extracted from growing mycelia using sterile acid-washed glass beads (Sigma, USA), as previously described [ ].

    Techniques: Polymerase Chain Reaction, Amplification, Southern Blot, Hybridization, Molecular Weight, Marker