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  • 99
    Thermo Fisher zero blunt topo pcr cloning kit
    Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in <t>PCR-TOPO</t> sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region
    Zero Blunt Topo Pcr Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7047 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi prism 7900ht real time pcr system
    SM induces the expression of proinflammatory cytokines/chemokines in lymph nodes and lungs. Animals were exposed to SM or vehicle, and total RNA was isolated from lymph node cells and lung tissues as described in Materials and Methods. The qPCR analysis was performed on the <t>ABI</t> <t>PRISM</t> <t>7900HT</t> Real-Time <t>PCR</t> System using the One-Step RT-PCR Master Mix. The relative expression of each mRNA was calculated in lymph node cells (A) and lungs (B).
    Abi Prism 7900ht Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 916 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Oxford Nanopore multiplex rt pcr amplicon sequencing
    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex <t>RT-PCR</t> <t>amplicon</t> sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Multiplex Rt Pcr Amplicon Sequencing, supplied by Oxford Nanopore, used in various techniques. Bioz Stars score: 99/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher abi prism 7500 sequence detection pcr system
    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex <t>RT-PCR</t> <t>amplicon</t> sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Abi Prism 7500 Sequence Detection Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcr 4 topo vector
    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex <t>RT-PCR</t> <t>amplicon</t> sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Pcr 4 Topo Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Pacific Biosciences pcr pacbio consensus sequences
    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex <t>RT-PCR</t> <t>amplicon</t> sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Pcr Pacbio Consensus Sequences, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 88/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc pcr primer sequences
    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is <t>PCR</t> amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an <t>Illumina</t> instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.
    Pcr Primer Sequences, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech pcr primer sequences
    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is <t>PCR</t> amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an <t>Illumina</t> instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.
    Pcr Primer Sequences, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 91/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher abi prism 7000 sequence detection pcr system
    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is <t>PCR</t> amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an <t>Illumina</t> instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.
    Abi Prism 7000 Sequence Detection Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher steponeplus sequence detection system
    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is <t>PCR</t> amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an <t>Illumina</t> instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.
    Steponeplus Sequence Detection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 7500 fast real time pcr sequence detection system
    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is <t>PCR</t> amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an <t>Illumina</t> instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.
    7500 Fast Real Time Pcr Sequence Detection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 3500xl genetic analyzer for sequence typing fragment analysis
    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is <t>PCR</t> amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an <t>Illumina</t> instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.
    3500xl Genetic Analyzer For Sequence Typing Fragment Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare sequenase pcr product sequence kit
    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is <t>PCR</t> amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an <t>Illumina</t> instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.
    Sequenase Pcr Product Sequence Kit, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcr product pre sequencing kit
    Cellular barcoding to appraise emergency DC development at a clonal level. (A) Experimental set up of cellular barcoding. Barcoded CD45.1 + CD11b − cKit + Sca1 + HSPCs were transplanted 3 d after sublethal irradiation (5 Gy) of CD45.2 recipient mice, followed by daily s.c. injection of PBS or Flt3L days 4-13. Splenic populations were isolated by FACS on day 14, lysed, and barcodes amplified by <t>PCR,</t> sequenced and analysed. (B) Possible explanations for emergency DC generation at a clonal level: 1) enhanced expansion of pre-existing HSPC clones; 2) recruitment of additional HSPC clones. (C) Total number of barcodes detected per recipient. (D) Number of barcodes present in each progeny splenic population. (E) t-SNE plots showing all barcoded clones (points), color depicts either fate cluster ID (left) or treatment (right). (F) Heatmap showing contribution to cell types (column) by individual barcodes (rows). Treatment and fate cluster ID of each <t>barcode</t> are annotated. (G) Number of barcodes present in each fate cluster. Data in (C-G) are pooled of five independent experiments; n = 14 PBS- (3,436 barcodes) and 14 Flt3L-treated (4,199 barcodes) mice. (C, D G) show mean ± SEM, P-values from two-tailed unpaired t-tests. See also Figure S4 for barcoding results using different experimental set up.
    Pcr Product Pre Sequencing Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Bioo Scientific nextflex pcr free dna sequencing kit
    The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid <t>DNA.</t> (A) Part of the Pcc CB plastid DNA was amplified by <t>PCR</t> from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.
    Nextflex Pcr Free Dna Sequencing Kit, supplied by Bioo Scientific, used in various techniques. Bioz Stars score: 89/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher 7900ht sequence detection pcr system
    The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid <t>DNA.</t> (A) Part of the Pcc CB plastid DNA was amplified by <t>PCR</t> from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.
    7900ht Sequence Detection Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 7300 real time pcr sequence detection system
    The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid <t>DNA.</t> (A) Part of the Pcc CB plastid DNA was amplified by <t>PCR</t> from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.
    7300 Real Time Pcr Sequence Detection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 7500 real time pcr system sequence detection software
    The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid <t>DNA.</t> (A) Part of the Pcc CB plastid DNA was amplified by <t>PCR</t> from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.
    7500 Real Time Pcr System Sequence Detection Software, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in PCR-TOPO sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region

    Journal: Nature Communications

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses

    doi: 10.1038/s41467-017-02706-7

    Figure Lengend Snippet: Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in PCR-TOPO sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region

    Article Snippet: Purified PCR products were cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci.

    Techniques: Recombinant, Introduce, Sequencing, Binding Assay, Polymerase Chain Reaction, Amplification

    SM induces the expression of proinflammatory cytokines/chemokines in lymph nodes and lungs. Animals were exposed to SM or vehicle, and total RNA was isolated from lymph node cells and lung tissues as described in Materials and Methods. The qPCR analysis was performed on the ABI PRISM 7900HT Real-Time PCR System using the One-Step RT-PCR Master Mix. The relative expression of each mRNA was calculated in lymph node cells (A) and lungs (B).

    Journal: International immunopharmacology

    Article Title: Sulfur Mustard Induces Immune Sensitization in Hairless Guinea Pigs

    doi: 10.1016/j.intimp.2009.10.015

    Figure Lengend Snippet: SM induces the expression of proinflammatory cytokines/chemokines in lymph nodes and lungs. Animals were exposed to SM or vehicle, and total RNA was isolated from lymph node cells and lung tissues as described in Materials and Methods. The qPCR analysis was performed on the ABI PRISM 7900HT Real-Time PCR System using the One-Step RT-PCR Master Mix. The relative expression of each mRNA was calculated in lymph node cells (A) and lungs (B).

    Article Snippet: The samples were resuspended in diethylpyrocarbonate (DEPC)-treated water (55°C for 10 min to dissolve RNA) and quantified spectrophotometrically. qPCR analysis was performed on the ABI PRISM 7900HT Real-Time PCR System using the One-Step RT-PCR Master Mix (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.

    Journal: Frontiers in Microbiology

    Article Title: Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

    doi: 10.3389/fmicb.2020.00514

    Figure Lengend Snippet: Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.

    Article Snippet: Quality Control and Comparison of Bioinformatic Pipelines In this study, the quality score of direct metatranscriptome RNA-seq was 83.4 and 83.7%, while 90.9 and 89.3% for multiplex RT-PCR amplicon sequencing from MinKNOW QC report ( ).

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Generated

    Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is PCR amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an Illumina instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.

    Journal: Nature protocols

    Article Title: PhIP-Seq Characterization of Serum Antibodies Using Oligonucleotide Encoded Peptidomes

    doi: 10.1038/s41596-018-0025-6

    Figure Lengend Snippet: Overview of the PhIP-seq methodology. Procedure step numbers are indicated in parentheses. A. A protein database is downloaded or designed. The pepsyn software is used to tile the protein sequences with overlapping peptide sequences. The oligonucleotide library encoding the peptide sequences is synthesized. The oligonucleotide library is PCR amplified with adapters for cloning into the phage display vector of choice. B. ELISA is used to quantify each sample’s IgG content for normalizing amount of antibody input into each phage binding reaction. Antibodies and their bound phage are captured using protein A/G coated magnetic beads. The library of peptide encoding DNA sequences are amplified by PCR directly from the immunoprecipitate. A second round of hemi-nested PCR is used to add sample-specific barcodes and sequencing adapters to the PCR1 product. Barcoded amplicons are pooled for sequencing on an Illumina instrument. C. Fastq sequencing files are demultiplexed and aligned to the reference sequences to obtain a count matrix. Statistical analysis of the count matrix is performed to determine peptide enrichments. Project specific analysis of peptide enrichments (e.g. identification of a common autoantigen) can then be carried out.

    Article Snippet: The PCR primer sequences presented here therefore include the Illumina sequencing adapters (suitable for both single and paired-end flow cells).

    Techniques: Software, Synthesized, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Binding Assay, Magnetic Beads, Nested PCR, Sequencing

    Cellular barcoding to appraise emergency DC development at a clonal level. (A) Experimental set up of cellular barcoding. Barcoded CD45.1 + CD11b − cKit + Sca1 + HSPCs were transplanted 3 d after sublethal irradiation (5 Gy) of CD45.2 recipient mice, followed by daily s.c. injection of PBS or Flt3L days 4-13. Splenic populations were isolated by FACS on day 14, lysed, and barcodes amplified by PCR, sequenced and analysed. (B) Possible explanations for emergency DC generation at a clonal level: 1) enhanced expansion of pre-existing HSPC clones; 2) recruitment of additional HSPC clones. (C) Total number of barcodes detected per recipient. (D) Number of barcodes present in each progeny splenic population. (E) t-SNE plots showing all barcoded clones (points), color depicts either fate cluster ID (left) or treatment (right). (F) Heatmap showing contribution to cell types (column) by individual barcodes (rows). Treatment and fate cluster ID of each barcode are annotated. (G) Number of barcodes present in each fate cluster. Data in (C-G) are pooled of five independent experiments; n = 14 PBS- (3,436 barcodes) and 14 Flt3L-treated (4,199 barcodes) mice. (C, D G) show mean ± SEM, P-values from two-tailed unpaired t-tests. See also Figure S4 for barcoding results using different experimental set up.

    Journal: bioRxiv

    Article Title: The clonal and molecular aetiology of emergency dendritic cell development

    doi: 10.1101/2020.05.28.120188

    Figure Lengend Snippet: Cellular barcoding to appraise emergency DC development at a clonal level. (A) Experimental set up of cellular barcoding. Barcoded CD45.1 + CD11b − cKit + Sca1 + HSPCs were transplanted 3 d after sublethal irradiation (5 Gy) of CD45.2 recipient mice, followed by daily s.c. injection of PBS or Flt3L days 4-13. Splenic populations were isolated by FACS on day 14, lysed, and barcodes amplified by PCR, sequenced and analysed. (B) Possible explanations for emergency DC generation at a clonal level: 1) enhanced expansion of pre-existing HSPC clones; 2) recruitment of additional HSPC clones. (C) Total number of barcodes detected per recipient. (D) Number of barcodes present in each progeny splenic population. (E) t-SNE plots showing all barcoded clones (points), color depicts either fate cluster ID (left) or treatment (right). (F) Heatmap showing contribution to cell types (column) by individual barcodes (rows). Treatment and fate cluster ID of each barcode are annotated. (G) Number of barcodes present in each fate cluster. Data in (C-G) are pooled of five independent experiments; n = 14 PBS- (3,436 barcodes) and 14 Flt3L-treated (4,199 barcodes) mice. (C, D G) show mean ± SEM, P-values from two-tailed unpaired t-tests. See also Figure S4 for barcoding results using different experimental set up.

    Article Snippet: Barcode amplification and sequencing PCR and sequencing were performed as described previously ( ).

    Techniques: Irradiation, Mouse Assay, Injection, Isolation, FACS, Amplification, Polymerase Chain Reaction, Two Tailed Test

    The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid DNA. (A) Part of the Pcc CB plastid DNA was amplified by PCR from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.

    Journal: PLoS ONE

    Article Title: The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi

    doi: 10.1371/journal.pone.0061778

    Figure Lengend Snippet: The rRNA/tRNA gene cluster of Plasmodium chabaudi chabaudi CB plastid DNA. (A) Part of the Pcc CB plastid DNA was amplified by PCR from total parasite DNA with the primers 1095 and 1096 (see Table 4 for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because rps4 and sufB share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of trnT , which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) ( trnR(ACG) *) and the 5' truncated rrl ( 'rrl ) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two trnT genes ( trnT -1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each trnT is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.

    Article Snippet: High Throughput Sequencing (HTS) Eight micrograms of total genomic DNA of Plasmodium chabaudi chabaudi isolate CB (Pcc CB) obtained from the blood of infected mice was fragmented in a microTUBE with an Adaptive Focused Acoustics fiber (Covaris, Woburn, Massachusetts) using an S2 focused ultra-sonicator (Covaris) set at 5% duty cycle; intensity 4; 200 cycles/burst for 90 s. In order to minimize the undesirable bias that affects the sequence analysis of extremely A/T-rich DNA such as that of Plasmodium spp., we processed the fragmented DNA using a NEXTflex PCR-Free DNA Sequencing Kit (BIOO Scientific, Austin, Texas) and NEXTflex PCR-Free barcode 1 (BIOO Scientific) following the protocol provided with the kit.

    Techniques: Periodic Counter-current Chromatography, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing