pcr amplicons Search Results


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  • 95
    New England Biolabs pcr amplicon
    Pcr Amplicon, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc pcr amplicons
    Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq <t>amplicons</t> span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. <t>PCR</t> fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.
    Pcr Amplicons, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Genewiz pcr amplicons
    Primers specific for OdAdV-1 were designed for a diagnostic <t>PCR</t> and a genotyping PCR. Assays were run using DNA extracted from additional diagnostic cases that were positive for OdAdV-1 or other viral agents to test for specificity. (a) The diagnostic PCR amplified a 502 bp amplicon, a 100 bp molecular weight marker, and OdAdV-1 from mule deer (lanes 1, 3), elk (lane 2), pronghorn (lanes 4, 8) and moose (lane 11). No amplification product was produced with samples positive for bovine, canine, or ovine adenovirus of undetermined type (lanes 5–7), chlamydophila from a mule deer (lane 9), or the no template control (lane 10). (b) The genotyping PCR was designed to span part of the variable region and produced <t>amplicons</t> of 680, 701, or 733 bp, depending on the presence or absence of deletions: lanes 1–4, California black-tailed deer (680 bp due to 53 bp deletion); lanes 5–8, pronghorn, white-tailed deer, mule deer, (701 bp due to 32 bp deletion); and lane 8–9, mule deer with the moose/elk variation (733 bp with no deletions). Traditional Sanger sequencing using forward and reverse primers confirmed the expected sequence changes.
    Pcr Amplicons, supplied by Genewiz, used in various techniques. Bioz Stars score: 93/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioTools Co polymerase chain reaction amplicons
    Primers specific for OdAdV-1 were designed for a diagnostic <t>PCR</t> and a genotyping PCR. Assays were run using DNA extracted from additional diagnostic cases that were positive for OdAdV-1 or other viral agents to test for specificity. (a) The diagnostic PCR amplified a 502 bp amplicon, a 100 bp molecular weight marker, and OdAdV-1 from mule deer (lanes 1, 3), elk (lane 2), pronghorn (lanes 4, 8) and moose (lane 11). No amplification product was produced with samples positive for bovine, canine, or ovine adenovirus of undetermined type (lanes 5–7), chlamydophila from a mule deer (lane 9), or the no template control (lane 10). (b) The genotyping PCR was designed to span part of the variable region and produced <t>amplicons</t> of 680, 701, or 733 bp, depending on the presence or absence of deletions: lanes 1–4, California black-tailed deer (680 bp due to 53 bp deletion); lanes 5–8, pronghorn, white-tailed deer, mule deer, (701 bp due to 32 bp deletion); and lane 8–9, mule deer with the moose/elk variation (733 bp with no deletions). Traditional Sanger sequencing using forward and reverse primers confirmed the expected sequence changes.
    Polymerase Chain Reaction Amplicons, supplied by BioTools Co, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher polymerase chain reaction pcr amplicons
    In vivo gene editing introduces a functional ORF in mdx 4cv mouse muscles. ( a ) Deep sequencing quantification on <t>PCR</t> <t>amplicons</t> generated from pooled genomic DNA extracted from muscles treated with strategy 1 (Δ5253, n =4), demonstrates successful gene editing at each of the individual target regions. Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (including insertions, deletions and substitutions), at sgRNA target sites in introns 51 and 53. ( b ) RT–PCR of target region transcripts isolated from TAs treated with strategy 1 (Δ5253, n =4) showing a predominant shorter product (red box), corresponding to approximately 87.5% of total transcripts based on image densitometry. ( c ) Subclone sequencing of the treatment-specific RT–PCR product (red box in b ) confirmed that these transcripts lacked the sequences encoded on exons 52 and 53 (the novel junction between exons 51 and 54 is highlighted in grey). ( d ) Deep sequencing quantification of gene editing efficiency on PCR amplicons generated from pooled genomic DNA (left, n =5) and RT–PCR amplicons generated from pooled transcripts (right, n =4) extracted from muscles treated with strategy 2 (53*). Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (red), HDR (white) or via a combination of both (black), at both sgRNA target sites in exon 53. ( e ) Deep sequencing reading frame analysis for strategy 2 (53*) shows the percentage of total edited transcript (gray) and genomic (black) reads resulting in frameshift indels, in-frame indels, in-frame deletions without the TAA stop codon (pΔ53), HDR reads (not including mixed NHEJ/HDR reads) and the total percentage of edited reads encoding a functional dystrophin ORF (HDR/pΔ53).
    Polymerase Chain Reaction Pcr Amplicons, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad pcr amplicons
    <t>PCR-single-strand</t> conformational polymorphism of the ovine IGF-1 gene. <t>Amplicons</t> were electrophoresed on a 12% non-denaturing acrylamide/bis-acrylamide gel; 200 V, 4 °C for 12 h. Three SSCP patterns, GG, AA and GA were detected.
    Pcr Amplicons, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 1143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Beckman Coulter pcr amplicons
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Pcr Amplicons, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 94/100, based on 1904 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Technelysium polymerase chain reaction amplicons
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Polymerase Chain Reaction Amplicons, supplied by Technelysium, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Pacific Biosciences polymerase chain reaction pcr amplicons
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Polymerase Chain Reaction Pcr Amplicons, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa polymerase chain reaction pcr amplicons
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Polymerase Chain Reaction Pcr Amplicons, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Transgenomic polymerase chain reaction pcr amplicons
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Polymerase Chain Reaction Pcr Amplicons, supplied by Transgenomic, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc polymerase chain reaction pcr amplicon libraries
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Polymerase Chain Reaction Pcr Amplicon Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc polymerase chain reaction pcr illumina amplicon sequencing protocol
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Polymerase Chain Reaction Pcr Illumina Amplicon Sequencing Protocol, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pcr amplicons
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Pcr Amplicons, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 2693 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Source BioScience plc pcr amplicons
    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates <t>amplicons</t> containing complementary ends. During <t>PCR</t> with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).
    Pcr Amplicons, supplied by Source BioScience plc, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Eurofins pcr amplicons
    CpG methylation profiles of TERT promoter proviral LTRs in selected tumor samples. Schematic representation of the CpG dinucleotide distribution in the ALV LTR is shown at the top. Bisulfite sequencing analysis of the LTR sequences are depicted as a linear array of open circles representing non-modified CpG residues and closed circles representing methylated CpG residues. Each line represents a representative sequenced result of the LTR region from host-proviral <t>PCR</t> <t>amplicons.</t> No methylation was detected in proviruses of tumor tissues tested.
    Pcr Amplicons, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    RainDance Technologies amplicon based microdroplet pcr
    CpG methylation profiles of TERT promoter proviral LTRs in selected tumor samples. Schematic representation of the CpG dinucleotide distribution in the ALV LTR is shown at the top. Bisulfite sequencing analysis of the LTR sequences are depicted as a linear array of open circles representing non-modified CpG residues and closed circles representing methylated CpG residues. Each line represents a representative sequenced result of the LTR region from host-proviral <t>PCR</t> <t>amplicons.</t> No methylation was detected in proviruses of tumor tissues tested.
    Amplicon Based Microdroplet Pcr, supplied by RainDance Technologies, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Oxford Nanopore 1d pcr barcoding amplicon kit
    CpG methylation profiles of TERT promoter proviral LTRs in selected tumor samples. Schematic representation of the CpG dinucleotide distribution in the ALV LTR is shown at the top. Bisulfite sequencing analysis of the LTR sequences are depicted as a linear array of open circles representing non-modified CpG residues and closed circles representing methylated CpG residues. Each line represents a representative sequenced result of the LTR region from host-proviral <t>PCR</t> <t>amplicons.</t> No methylation was detected in proviruses of tumor tissues tested.
    1d Pcr Barcoding Amplicon Kit, supplied by Oxford Nanopore, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Innogenetics gene specific pcr amplicons
    CpG methylation profiles of TERT promoter proviral LTRs in selected tumor samples. Schematic representation of the CpG dinucleotide distribution in the ALV LTR is shown at the top. Bisulfite sequencing analysis of the LTR sequences are depicted as a linear array of open circles representing non-modified CpG residues and closed circles representing methylated CpG residues. Each line represents a representative sequenced result of the LTR region from host-proviral <t>PCR</t> <t>amplicons.</t> No methylation was detected in proviruses of tumor tissues tested.
    Gene Specific Pcr Amplicons, supplied by Innogenetics, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.

    Journal: PLoS Biology

    Article Title: Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

    doi: 10.1371/journal.pbio.2005595

    Figure Lengend Snippet: Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.

    Article Snippet: Editing was quantified by Illumina sequencing of PCR amplicons spanning both the site of cleavage and an allelic SNP ( and , for detailed experimental protocols see ).

    Techniques: Mutagenesis, High Throughput Screening Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Mouse Assay, CpG Methylation Assay, Non-Homologous End Joining

    Primers specific for OdAdV-1 were designed for a diagnostic PCR and a genotyping PCR. Assays were run using DNA extracted from additional diagnostic cases that were positive for OdAdV-1 or other viral agents to test for specificity. (a) The diagnostic PCR amplified a 502 bp amplicon, a 100 bp molecular weight marker, and OdAdV-1 from mule deer (lanes 1, 3), elk (lane 2), pronghorn (lanes 4, 8) and moose (lane 11). No amplification product was produced with samples positive for bovine, canine, or ovine adenovirus of undetermined type (lanes 5–7), chlamydophila from a mule deer (lane 9), or the no template control (lane 10). (b) The genotyping PCR was designed to span part of the variable region and produced amplicons of 680, 701, or 733 bp, depending on the presence or absence of deletions: lanes 1–4, California black-tailed deer (680 bp due to 53 bp deletion); lanes 5–8, pronghorn, white-tailed deer, mule deer, (701 bp due to 32 bp deletion); and lane 8–9, mule deer with the moose/elk variation (733 bp with no deletions). Traditional Sanger sequencing using forward and reverse primers confirmed the expected sequence changes.

    Journal: The Journal of General Virology

    Article Title: Whole-genome sequences of Odocoileus hemionus deer adenovirus isolates from deer, moose and elk are highly conserved and support a new species in the genus Atadenovirus

    doi: 10.1099/jgv.0.000880

    Figure Lengend Snippet: Primers specific for OdAdV-1 were designed for a diagnostic PCR and a genotyping PCR. Assays were run using DNA extracted from additional diagnostic cases that were positive for OdAdV-1 or other viral agents to test for specificity. (a) The diagnostic PCR amplified a 502 bp amplicon, a 100 bp molecular weight marker, and OdAdV-1 from mule deer (lanes 1, 3), elk (lane 2), pronghorn (lanes 4, 8) and moose (lane 11). No amplification product was produced with samples positive for bovine, canine, or ovine adenovirus of undetermined type (lanes 5–7), chlamydophila from a mule deer (lane 9), or the no template control (lane 10). (b) The genotyping PCR was designed to span part of the variable region and produced amplicons of 680, 701, or 733 bp, depending on the presence or absence of deletions: lanes 1–4, California black-tailed deer (680 bp due to 53 bp deletion); lanes 5–8, pronghorn, white-tailed deer, mule deer, (701 bp due to 32 bp deletion); and lane 8–9, mule deer with the moose/elk variation (733 bp with no deletions). Traditional Sanger sequencing using forward and reverse primers confirmed the expected sequence changes.

    Article Snippet: Sanger sequencing of the PCR amplicons using forward and reverse primers (GENEWIZ, South Plainfield, NJ, USA) was used to verify the amplicons.

    Techniques: Diagnostic Assay, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Produced, Sequencing

    In vivo gene editing introduces a functional ORF in mdx 4cv mouse muscles. ( a ) Deep sequencing quantification on PCR amplicons generated from pooled genomic DNA extracted from muscles treated with strategy 1 (Δ5253, n =4), demonstrates successful gene editing at each of the individual target regions. Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (including insertions, deletions and substitutions), at sgRNA target sites in introns 51 and 53. ( b ) RT–PCR of target region transcripts isolated from TAs treated with strategy 1 (Δ5253, n =4) showing a predominant shorter product (red box), corresponding to approximately 87.5% of total transcripts based on image densitometry. ( c ) Subclone sequencing of the treatment-specific RT–PCR product (red box in b ) confirmed that these transcripts lacked the sequences encoded on exons 52 and 53 (the novel junction between exons 51 and 54 is highlighted in grey). ( d ) Deep sequencing quantification of gene editing efficiency on PCR amplicons generated from pooled genomic DNA (left, n =5) and RT–PCR amplicons generated from pooled transcripts (right, n =4) extracted from muscles treated with strategy 2 (53*). Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (red), HDR (white) or via a combination of both (black), at both sgRNA target sites in exon 53. ( e ) Deep sequencing reading frame analysis for strategy 2 (53*) shows the percentage of total edited transcript (gray) and genomic (black) reads resulting in frameshift indels, in-frame indels, in-frame deletions without the TAA stop codon (pΔ53), HDR reads (not including mixed NHEJ/HDR reads) and the total percentage of edited reads encoding a functional dystrophin ORF (HDR/pΔ53).

    Journal: Nature Communications

    Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

    doi: 10.1038/ncomms14454

    Figure Lengend Snippet: In vivo gene editing introduces a functional ORF in mdx 4cv mouse muscles. ( a ) Deep sequencing quantification on PCR amplicons generated from pooled genomic DNA extracted from muscles treated with strategy 1 (Δ5253, n =4), demonstrates successful gene editing at each of the individual target regions. Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (including insertions, deletions and substitutions), at sgRNA target sites in introns 51 and 53. ( b ) RT–PCR of target region transcripts isolated from TAs treated with strategy 1 (Δ5253, n =4) showing a predominant shorter product (red box), corresponding to approximately 87.5% of total transcripts based on image densitometry. ( c ) Subclone sequencing of the treatment-specific RT–PCR product (red box in b ) confirmed that these transcripts lacked the sequences encoded on exons 52 and 53 (the novel junction between exons 51 and 54 is highlighted in grey). ( d ) Deep sequencing quantification of gene editing efficiency on PCR amplicons generated from pooled genomic DNA (left, n =5) and RT–PCR amplicons generated from pooled transcripts (right, n =4) extracted from muscles treated with strategy 2 (53*). Shown are the percentages of total reads that displayed genomic modifications occurring as a result of NHEJ (red), HDR (white) or via a combination of both (black), at both sgRNA target sites in exon 53. ( e ) Deep sequencing reading frame analysis for strategy 2 (53*) shows the percentage of total edited transcript (gray) and genomic (black) reads resulting in frameshift indels, in-frame indels, in-frame deletions without the TAA stop codon (pΔ53), HDR reads (not including mixed NHEJ/HDR reads) and the total percentage of edited reads encoding a functional dystrophin ORF (HDR/pΔ53).

    Article Snippet: Approximately 500 bp amplicons across the targeted regions of genomic DNA were generated by PCR using Phusion proof-reading polymerase (New England Biolabs, NEB) and analysed for targeting efficiency using T7 endonuclease 1 (NEB), next generation sequencing (BGI International or in-house) or Sanger sequencing (Simpleseq, Eurofins MWG Operon) of subclones of PCR amplicons (Zero Blunt TOPO, Invitrogen).

    Techniques: In Vivo, Functional Assay, Sequencing, Polymerase Chain Reaction, Generated, Non-Homologous End Joining, Reverse Transcription Polymerase Chain Reaction, Isolation

    PCR-single-strand conformational polymorphism of the ovine IGF-1 gene. Amplicons were electrophoresed on a 12% non-denaturing acrylamide/bis-acrylamide gel; 200 V, 4 °C for 12 h. Three SSCP patterns, GG, AA and GA were detected.

    Journal: Journal of Genetic Engineering & Biotechnology

    Article Title: New polymorphism in the 5′ flanking region of IGF-1 gene and its association with wool traits in Egyptian Barki sheep

    doi: 10.1016/j.jgeb.2017.08.001

    Figure Lengend Snippet: PCR-single-strand conformational polymorphism of the ovine IGF-1 gene. Amplicons were electrophoresed on a 12% non-denaturing acrylamide/bis-acrylamide gel; 200 V, 4 °C for 12 h. Three SSCP patterns, GG, AA and GA were detected.

    Article Snippet: PCR amplicons were electrophoresed in 2% agarose gels, using 0.5X TBE buffer (89 mM Tris, 89 mM boric acid and 2 mM Na2 EDTA) containing 200 ng/ml of ethidium bromide and was visualized under UV light and photographed by Bio-Rad Laboratories, Hercules, CA, USA.

    Techniques: Polymerase Chain Reaction, Acrylamide Gel Assay

    Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates amplicons containing complementary ends. During PCR with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).

    Journal: Nucleic Acids Research

    Article Title: Whole transcriptome profiling reveals the RNA content of motor axons

    doi: 10.1093/nar/gkv1027

    Figure Lengend Snippet: Optimization of double-random priming and amplification efficiency. ( A ) Schematic outline of the double-random priming strategy for cDNA amplification. RNA (red) is reverse-transcribed using an oligonucleotide adapter (blue) containing random octamers. Second strand synthesis using the same primer generates amplicons containing complementary ends. During PCR with the adapter primer amplification of short fragments is suppressed due to formation of panhandle-like structures preventing primer annealing. Only longer amplicons of sufficient size are amplified. ( B ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Gapdh levels were measured by qPCR at various timepoints during the PCR amplification. The legend describes the variables tested as following: polymerase used during second strand synthesis/final primer concentration in μM for second strand synthesis/final primer concentration in μM for PCR amplification. Ct, crossing point. ( C ) Whole transcriptome amplification efficiency for different polymerases and primer concentrations. Ubqln2 levels were measured by qPCR. The legend is configured as in (B).

    Article Snippet: PCR amplicons were purified with AMPure XP beads (Beckman Coulter).

    Techniques: Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Concentration Assay

    CpG methylation profiles of TERT promoter proviral LTRs in selected tumor samples. Schematic representation of the CpG dinucleotide distribution in the ALV LTR is shown at the top. Bisulfite sequencing analysis of the LTR sequences are depicted as a linear array of open circles representing non-modified CpG residues and closed circles representing methylated CpG residues. Each line represents a representative sequenced result of the LTR region from host-proviral PCR amplicons. No methylation was detected in proviruses of tumor tissues tested.

    Journal: Viruses

    Article Title: ALV Integration-Associated Hypomethylation at the TERT Promoter Locus

    doi: 10.3390/v10020074

    Figure Lengend Snippet: CpG methylation profiles of TERT promoter proviral LTRs in selected tumor samples. Schematic representation of the CpG dinucleotide distribution in the ALV LTR is shown at the top. Bisulfite sequencing analysis of the LTR sequences are depicted as a linear array of open circles representing non-modified CpG residues and closed circles representing methylated CpG residues. Each line represents a representative sequenced result of the LTR region from host-proviral PCR amplicons. No methylation was detected in proviruses of tumor tissues tested.

    Article Snippet: PCR amplicons were analyzed by conventional sequencing provided by Eurofins Genomics services (Louisville, KY, USA).

    Techniques: CpG Methylation Assay, Methylation Sequencing, Modification, Methylation, Polymerase Chain Reaction