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Illumina Inc pcr primers
<t>PCR</t> amplification, quantitative PCR and Illumina sequencing schema . (a) Diagrammatic representation of the complete integrated shRNA construct. LTR, long terminal repeat; Ze, zeomycin resistance bacterial selectable marker; tGFP, turbo GFP; IRES, internal ribosome entry site; Puro, puromycin mammalian selectable marker; RRE, Rev response element; sinLT, self-interacting LTR. (b) The structure of the shRNAmir construct. The sense and antisense shRNA sequences hybridize to form a hairpin loop structure. (c) PCR primer alignment to the shRNA construct. The PCR primers incorporate p7 and <t>p5</t> sequences to enable capture on an Illumina flowcell. (d) Sequencing primer, quantitative PCR (qPCR) primer and qPCR dual label probe alignment to the shRNA PCR product. CMV, cytomegalovirus; DLP, dual-labeled probe.
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1) Product Images from "High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing"

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

Journal: Genome Biology

doi: 10.1186/gb-2011-12-10-r104

PCR amplification, quantitative PCR and Illumina sequencing schema . (a) Diagrammatic representation of the complete integrated shRNA construct. LTR, long terminal repeat; Ze, zeomycin resistance bacterial selectable marker; tGFP, turbo GFP; IRES, internal ribosome entry site; Puro, puromycin mammalian selectable marker; RRE, Rev response element; sinLT, self-interacting LTR. (b) The structure of the shRNAmir construct. The sense and antisense shRNA sequences hybridize to form a hairpin loop structure. (c) PCR primer alignment to the shRNA construct. The PCR primers incorporate p7 and p5 sequences to enable capture on an Illumina flowcell. (d) Sequencing primer, quantitative PCR (qPCR) primer and qPCR dual label probe alignment to the shRNA PCR product. CMV, cytomegalovirus; DLP, dual-labeled probe.
Figure Legend Snippet: PCR amplification, quantitative PCR and Illumina sequencing schema . (a) Diagrammatic representation of the complete integrated shRNA construct. LTR, long terminal repeat; Ze, zeomycin resistance bacterial selectable marker; tGFP, turbo GFP; IRES, internal ribosome entry site; Puro, puromycin mammalian selectable marker; RRE, Rev response element; sinLT, self-interacting LTR. (b) The structure of the shRNAmir construct. The sense and antisense shRNA sequences hybridize to form a hairpin loop structure. (c) PCR primer alignment to the shRNA construct. The PCR primers incorporate p7 and p5 sequences to enable capture on an Illumina flowcell. (d) Sequencing primer, quantitative PCR (qPCR) primer and qPCR dual label probe alignment to the shRNA PCR product. CMV, cytomegalovirus; DLP, dual-labeled probe.

Techniques Used: Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Sequencing, shRNA, Construct, Marker, Labeling

Quantitative PCR quantification of PCR products . Quantitative PCR (qPCR) assay designed to detect and quantify all amplifiable solexa molecules (using oligos p5/p7 and SybrGreen) or shRNA-specific PCR products (using Taqman, amplification primers p5/p7 and a dual-labeled probe). (a) shRNA PCR products quantified against a library of known concentration. (b) Standard curve constructed using a ten-fold dilution series covering 100, 10, 1 and 0.1 pM. (c) Agilent electrophoresis profile of reference library.
Figure Legend Snippet: Quantitative PCR quantification of PCR products . Quantitative PCR (qPCR) assay designed to detect and quantify all amplifiable solexa molecules (using oligos p5/p7 and SybrGreen) or shRNA-specific PCR products (using Taqman, amplification primers p5/p7 and a dual-labeled probe). (a) shRNA PCR products quantified against a library of known concentration. (b) Standard curve constructed using a ten-fold dilution series covering 100, 10, 1 and 0.1 pM. (c) Agilent electrophoresis profile of reference library.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, shRNA, Amplification, Labeling, Concentration Assay, Construct, Electrophoresis

2) Product Images from "The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms"

Article Title: The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01692-15

Ordination plots of bacterial community structures as determined by nonmetric multidimensional scaling (nMDS) of PCR-Illumina profiles of 16S rRNA gene fragments in the BAC filters at SPRWS. (A) Bacterial community dynamics in filter 3. (B) Bacterial community dynamics in filter 6. (C) Bacterial community dynamics in filter 13. (D) Comparison of bacterial community structures in different filters on 1 July 2011; different filters are represented by a number. ●, 15 March 2011; ■, 31 May 2011; ▲, 1 July 2011; ◆, 11 August 2011; ○, 22 November 2011; □, 15 December 2011; △, 14 February 2012. The data are shown as the arithmetic means of triplicate profiles (±1 standard deviation).
Figure Legend Snippet: Ordination plots of bacterial community structures as determined by nonmetric multidimensional scaling (nMDS) of PCR-Illumina profiles of 16S rRNA gene fragments in the BAC filters at SPRWS. (A) Bacterial community dynamics in filter 3. (B) Bacterial community dynamics in filter 6. (C) Bacterial community dynamics in filter 13. (D) Comparison of bacterial community structures in different filters on 1 July 2011; different filters are represented by a number. ●, 15 March 2011; ■, 31 May 2011; ▲, 1 July 2011; ◆, 11 August 2011; ○, 22 November 2011; □, 15 December 2011; △, 14 February 2012. The data are shown as the arithmetic means of triplicate profiles (±1 standard deviation).

Techniques Used: Polymerase Chain Reaction, BAC Assay, Standard Deviation

Comparison of the relative quantities of ammonia-oxidizing and nitrite-oxidizing bacteria in three SPRWS BAC filters. The values for AOB (Illumina) and NOB (Illumina) represent the fractions of known AOB and Nitrospira sequences, respectively, found in an Illumina profile divided by the total number of sequences in that profile. The amoA /16S rRNA represents the ratio of amoA to 16S rRNA genes as measured by quantitative real-time PCR (see Data Sets S2 to S4 in the supplemental material).
Figure Legend Snippet: Comparison of the relative quantities of ammonia-oxidizing and nitrite-oxidizing bacteria in three SPRWS BAC filters. The values for AOB (Illumina) and NOB (Illumina) represent the fractions of known AOB and Nitrospira sequences, respectively, found in an Illumina profile divided by the total number of sequences in that profile. The amoA /16S rRNA represents the ratio of amoA to 16S rRNA genes as measured by quantitative real-time PCR (see Data Sets S2 to S4 in the supplemental material).

Techniques Used: BAC Assay, Real-time Polymerase Chain Reaction

3) Product Images from "Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites"

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites

Journal: Human Gene Therapy Methods

doi: 10.1089/hgtb.2015.060

Illumina MiSeq MGS-PCR. Genomic DNA is sheared, resulting in smaller genomic fragments that may contain the vector provirus. Fragments are blunt ended and linker cassettes ligated to the ends of the fragment. Thirty rounds of exponential PCR are conducted
Figure Legend Snippet: Illumina MiSeq MGS-PCR. Genomic DNA is sheared, resulting in smaller genomic fragments that may contain the vector provirus. Fragments are blunt ended and linker cassettes ligated to the ends of the fragment. Thirty rounds of exponential PCR are conducted

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

Illumina MiSeq universal primers. The Illumina universal primers (forward and reverse) consist of an adapter, indices, stem, and the user-defined specific primer sequences. In our adaptation for MGS-PCR, the forward primer is designed for the LTR, while
Figure Legend Snippet: Illumina MiSeq universal primers. The Illumina universal primers (forward and reverse) consist of an adapter, indices, stem, and the user-defined specific primer sequences. In our adaptation for MGS-PCR, the forward primer is designed for the LTR, while

Techniques Used: Polymerase Chain Reaction

4) Product Images from "pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping"

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw1261

Construction of pBACode cloning vectors. ( A ) A pBACode-1 pool is generated by PCR amplification using pcc2FOS as a template. The 3΄ ends of primers are complementary to the vector sequences flanking the cloning sites so that the whole pcc2FOS vector except for its cloning sites is amplified. The 5΄ ends of the primers carry the cloning sites followed by 20-bp random sequences. ( B ) A pBACode-2 pool is generated by a combination of PCR amplification and subcloning. First, an intermediate vector is constructed based on pcc2FOS such that its lacZ sequence is replaced by an I-SceI site. The second intermediate vector is constructed by inserting the kan R selection gene marker into the cloning site of pcc2FOS, and then the lacZ-kan R -lacZ segment is amplified using primers containing random barcodes. The resulting PCR product is subcloned into the first intermediate vector. Kanamycin selection is used to eliminate no-insert vectors.
Figure Legend Snippet: Construction of pBACode cloning vectors. ( A ) A pBACode-1 pool is generated by PCR amplification using pcc2FOS as a template. The 3΄ ends of primers are complementary to the vector sequences flanking the cloning sites so that the whole pcc2FOS vector except for its cloning sites is amplified. The 5΄ ends of the primers carry the cloning sites followed by 20-bp random sequences. ( B ) A pBACode-2 pool is generated by a combination of PCR amplification and subcloning. First, an intermediate vector is constructed based on pcc2FOS such that its lacZ sequence is replaced by an I-SceI site. The second intermediate vector is constructed by inserting the kan R selection gene marker into the cloning site of pcc2FOS, and then the lacZ-kan R -lacZ segment is amplified using primers containing random barcodes. The resulting PCR product is subcloned into the first intermediate vector. Kanamycin selection is used to eliminate no-insert vectors.

Techniques Used: Clone Assay, Generated, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Subcloning, Construct, Sequencing, Selection, Marker

5) Product Images from "Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir"

Article Title: Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir

Journal: Environmental Microbiology

doi: 10.1111/1462-2920.13706

Taxonomic summary of the McElmo Dome microbial community constructed using RDP/Silva‐annotated 16S rRNA gene sequencing of clone and Illumina (NGS) libraries, and Illumina binned metagenome (MG) read frequencies on the (A) phylum and (B) genus level. K, P, pK, and pP refer to methods of DNA extraction and PCR template preparation, as described in experimental procedures (K = kit extracted gDNA, P = phenol extracted gDNA, p = clone library vector with 16S rRNA amplicon used as template).
Figure Legend Snippet: Taxonomic summary of the McElmo Dome microbial community constructed using RDP/Silva‐annotated 16S rRNA gene sequencing of clone and Illumina (NGS) libraries, and Illumina binned metagenome (MG) read frequencies on the (A) phylum and (B) genus level. K, P, pK, and pP refer to methods of DNA extraction and PCR template preparation, as described in experimental procedures (K = kit extracted gDNA, P = phenol extracted gDNA, p = clone library vector with 16S rRNA amplicon used as template).

Techniques Used: Construct, Sequencing, Next-Generation Sequencing, DNA Extraction, Polymerase Chain Reaction, Plasmid Preparation, Amplification

6) Product Images from "Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis"

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis

Journal: BMC Genomics

doi: 10.1186/1471-2164-12-628

Identification of introns . (A) Non strand-specific RNA-seq (data is from YPD media at 30°C) showing the expression level for gene Cpar2_601470. Although expression of the 3' UTR region is low, two potential introns were identified. The red arrows show the location of oligonucleotide primers designed to confirm the presence of the introns. (B) Intron validation by PCR. Pairs of oligonucleotide primers flanking the intronic region were used to amplify products from genomic DNA (D) or cDNA (R). Introns were confirmed in the 3' UTR of Cpar2_601470, the 5' UTRs of Cpar2_400980 and Cpar2_205880, and within the coding sequence of 5 other genes (details of expected sizes are shown in Additional file 8 ). The small bands in Cpar2_601830 and Cpar2_807560 are likely due to the presence of excess primers or to nonspecific amplification.
Figure Legend Snippet: Identification of introns . (A) Non strand-specific RNA-seq (data is from YPD media at 30°C) showing the expression level for gene Cpar2_601470. Although expression of the 3' UTR region is low, two potential introns were identified. The red arrows show the location of oligonucleotide primers designed to confirm the presence of the introns. (B) Intron validation by PCR. Pairs of oligonucleotide primers flanking the intronic region were used to amplify products from genomic DNA (D) or cDNA (R). Introns were confirmed in the 3' UTR of Cpar2_601470, the 5' UTRs of Cpar2_400980 and Cpar2_205880, and within the coding sequence of 5 other genes (details of expected sizes are shown in Additional file 8 ). The small bands in Cpar2_601830 and Cpar2_807560 are likely due to the presence of excess primers or to nonspecific amplification.

Techniques Used: RNA Sequencing Assay, Expressing, Polymerase Chain Reaction, Sequencing, Amplification

Related Articles

Clone Assay:

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: BAC end sequencing and local assembly BAC clones were cultured in 96-well plates before mixing and DNA extraction. .. The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences.

Amplification:

Article Title: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs
Article Snippet: PCR amplification was performed using Accuprime Supermix I (Life Technologies 12342028) and 20 to 24 cycles to avoid the formation of secondary products. .. PCR primers contained the Illumina P5 and P3 sequences together with degenerated barcodes for PCR duplicates removal.

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
Article Snippet: .. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR. .. All libraries were quantified using a Qubit Fluorometer (Invitrogen) and assessed on a 2% agarose gel.

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing
Article Snippet: To facilitate this we used PCR amplification of genomic DNA from screen cell populations, based upon methods previously described by McManus [ ], Hannon, Elledge and Lowe [ , , ] and Quail [ ]. .. The PCR primers also encompassed p5 and p7 sequences that allow sequence capture and sequencing-by-synthesis on the Illumina GAIIx platform.

Article Title: Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila
Article Snippet: The PCR primers contained overhangs so that Illumina clustering, indexing, and sequencing oligonucleotides could be added in a subsequent nested PCR. .. The same primers designed for ratchet point lariat amplification were also used in different combinations to attempt to amplify the branchpoint lariats that would be generated by skipping one or more ratchet points.

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: The circularized DNA was used as a template for inverse PCR amplification of BAC ends. .. The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences.

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: CRISPR arrays were amplified by PCR using a set of primers in which the forward primer annealed within the leader region of the CRISPR array and the reverse primer annealed to the existing spacer closest to the leader (referred to as the first round of PCR). .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing.

Article Title: Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy
Article Snippet: PCR primers included Illumina TruSeq sequencing primer sequences on the 5′ ends ( ). .. Because PCR products averaged 106 bp in length, the 3′ ends of paired reads overlapped, and that overlap was used to infer complete amplicon fragments via single ungapped alignment, parameterized to score each match as 5 and each mismatch as −4.

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: After single-cell genome amplification with a MALBAC Single Cell WGA Kit (Yikon Genomics), target regions of genes of interest were PCR-amplified with primers including well indexes by PCR. .. PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences.

Whole Genome Amplification:

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: After single-cell genome amplification with a MALBAC Single Cell WGA Kit (Yikon Genomics), target regions of genes of interest were PCR-amplified with primers including well indexes by PCR. .. PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences.

Construct:

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing
Article Snippet: PCR primers complementary to constant regions found in all shRNA constructs (Figure ) were used to amplify the shRNA target sequence that is specific to each individual shRNA construct. .. The PCR primers also encompassed p5 and p7 sequences that allow sequence capture and sequencing-by-synthesis on the Illumina GAIIx platform.

Article Title: Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir
Article Snippet: Phylogenetic tree was constructed with 100X bootstrapping. .. Summary of pcr primers used in clone library and Illumina sequencing preparation.

Modification:

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites
Article Snippet: .. For adaptation, modified nested PCR primers including the Illumina universal adapters, indices, and stems for MiSeq paired-end sequencing were prepared ( ). .. We performed Illumina MiSeq paired-end sequencing on MGS-PCR samples that had expanded clones, or were oligoclonal, or highly polyclonal: (1) lentiviral vector-mutagenized orthotopic primary prostate tumors that had expanded clones, (2) gammaretroviral vector-mutagenized CD105+ , Sca-1+ -enriched mouse bone marrow that was expanded in vitro and was oligoclonal, and (3) foamy viral vector-mutagenized human CD34+ cells that were highly polyclonal.

Transformation Assay:

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: Spacer acquisition assay and high throughput sequencing Twenty colonies of P. furiosus strains transformed with plasmid were inoculated in 5 ml defined medium containing 20 μM uracil. .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing.

Hybridization:

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
Article Snippet: Illumina adapters were ligated to the ends of the DNA fragments, allowing for the subsequent hybridization to a flow cell. .. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR.

Flow Cytometry:

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
Article Snippet: Illumina adapters were ligated to the ends of the DNA fragments, allowing for the subsequent hybridization to a flow cell. .. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR.

Inverse PCR:

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: The circularized DNA was used as a template for inverse PCR amplification of BAC ends. .. The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences.

Genomic Sequencing:

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences. .. We extracted barcodes and genomic sequences from read pairs using in-house Perl scripts.

Cell Culture:

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: BAC end sequencing and local assembly BAC clones were cultured in 96-well plates before mixing and DNA extraction. .. The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences.

Generated:

Article Title: Heteroplasmic mitochondrial DNA mutations in normal and tumor cells
Article Snippet: Determination of background error rates PCR products used for Genome Analyzer II sequencing were generated through two PCR steps. .. In the second step PCR, one pair of PCR primers were designed to contain the universal sequence used in the first step and the Illumina grafting primers (Forward Primer: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; Reverse Primer: 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3′).

Article Title: Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila
Article Snippet: The PCR primers contained overhangs so that Illumina clustering, indexing, and sequencing oligonucleotides could be added in a subsequent nested PCR. .. The same primers designed for ratchet point lariat amplification were also used in different combinations to attempt to amplify the branchpoint lariats that would be generated by skipping one or more ratchet points.

Sequencing:

Article Title: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs
Article Snippet: PCR primers contained the Illumina P5 and P3 sequences together with degenerated barcodes for PCR duplicates removal. .. The iCLIP libraries were sequenced on a NextSeq 500 using 75-nt single-read sequencing runs.

Article Title: Heteroplasmic mitochondrial DNA mutations in normal and tumor cells
Article Snippet: .. In the second step PCR, one pair of PCR primers were designed to contain the universal sequence used in the first step and the Illumina grafting primers (Forward Primer: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; Reverse Primer: 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3′). .. The PCR was conducted in a single PCR tube using this pair of primers and the diluted PCR product mixture from first step as template.

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
Article Snippet: In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR. .. Sequencing was carried out in-house by running at least 36 cycles on an Illumina Genome Analyzer IIx according to manufacturer's instructions, resulting in read lengths of approximately 42 bases.

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing
Article Snippet: .. The PCR primers also encompassed p5 and p7 sequences that allow sequence capture and sequencing-by-synthesis on the Illumina GAIIx platform. .. To enable sufficient representation of each shRNA in the screening pool, multiple PCR reactions were performed in parallel to generate the sequencing library from each shRNA pool.

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites
Article Snippet: .. For adaptation, modified nested PCR primers including the Illumina universal adapters, indices, and stems for MiSeq paired-end sequencing were prepared ( ). .. We performed Illumina MiSeq paired-end sequencing on MGS-PCR samples that had expanded clones, or were oligoclonal, or highly polyclonal: (1) lentiviral vector-mutagenized orthotopic primary prostate tumors that had expanded clones, (2) gammaretroviral vector-mutagenized CD105+ , Sca-1+ -enriched mouse bone marrow that was expanded in vitro and was oligoclonal, and (3) foamy viral vector-mutagenized human CD34+ cells that were highly polyclonal.

Article Title: Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila
Article Snippet: .. The PCR primers contained overhangs so that Illumina clustering, indexing, and sequencing oligonucleotides could be added in a subsequent nested PCR. .. The same primers designed for ratchet point lariat amplification were also used in different combinations to attempt to amplify the branchpoint lariats that would be generated by skipping one or more ratchet points.

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: Paragraph title: BAC end sequencing and local assembly ... The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences.

Article Title: The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms
Article Snippet: Analysis of publically available 16S rRNA sequences from Nitrosomonas spp., however, confirmed 100% sequence identity with the PCR primers used in this study. .. However, we have previously used this DNA extraction procedure in combination with the PCR primers targeting the V3 region of the 16S rRNA gene (absent the Illumina adapters and barcodes) to characterize a nitrifying enrichment culture in which Nitrosomonas spp. comprised as much as 25 to 70% of the bacterial community , again suggesting that analytical biases against the AOB of the order Nitrosomonadales are unlikely.

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing. ..

Article Title: Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy
Article Snippet: .. PCR primers included Illumina TruSeq sequencing primer sequences on the 5′ ends ( ). .. A second round of PCR was then used to add Illumina flowcell binding sequences and experiment-specific indexes 5′ of the primer sequence.

Article Title: Genome-Wide Profiling of Cap-Independent Translation Enhancing Elements in the Human Genome
Article Snippet: Paragraph title: Sequence analysis ... To detect and trim the PCR primers at both ends of each Illumina read, we used the “cutadapt” program ( http://code.google.com/p/cutadapt/ ) allowing a maximum of 2 mismatches.

Article Title: Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir
Article Snippet: .. Summary of pcr primers used in clone library and Illumina sequencing preparation. ..

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences. .. In this study, variation genotype was assigned on assumption that sequencing error rate is lower than 2% and apparent variation with allele frequency lower than 2% was regarded as wild-type.

Binding Assay:

Article Title: Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy
Article Snippet: PCR primers included Illumina TruSeq sequencing primer sequences on the 5′ ends ( ). .. A second round of PCR was then used to add Illumina flowcell binding sequences and experiment-specific indexes 5′ of the primer sequence.

DNA Extraction:

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: BAC end sequencing and local assembly BAC clones were cultured in 96-well plates before mixing and DNA extraction. .. The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences.

Article Title: The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms
Article Snippet: .. However, we have previously used this DNA extraction procedure in combination with the PCR primers targeting the V3 region of the 16S rRNA gene (absent the Illumina adapters and barcodes) to characterize a nitrifying enrichment culture in which Nitrosomonas spp. comprised as much as 25 to 70% of the bacterial community , again suggesting that analytical biases against the AOB of the order Nitrosomonadales are unlikely. .. A second (but much weaker) line of evidence supporting the claim that novel ammonia-oxidizing organisms are present in the BAC filters is the exceptionally high ratio of NOB to AOB (NOB/AOB ratio of 225) in the Illumina MiSeq profiles.

Nucleic Acid Electrophoresis:

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: These larger, expanded PCR products were separated from unexpanded products by gel electrophoresis followed by DNA recovery (Zymo Research). .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing.

RNA Sequencing Assay:

Article Title: Transcriptome Sequencing Identifies PCAT-1, a Novel lincRNA Implicated in Prostate Cancer Progression
Article Snippet: .. RNA-Seq library preparation 2μg total RNA was selected for polyA+ RNA using Sera-Mag oligo(dT) beads (Thermo Scientific), and paired-end next-generation sequencing libraries were prepared as previously described using Illumina-supplied universal adaptor oligos and PCR primers (Illumina). .. Samples were sequenced in a single lane on an Illumina Genome Analyzer I or Genome Analyzer II flowcell using previously described protocols.

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
Article Snippet: Paragraph title: RNA-seq library preparation ... In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR.

Mutagenesis:

Article Title: Heteroplasmic mitochondrial DNA mutations in normal and tumor cells
Article Snippet: In the second step PCR, one pair of PCR primers were designed to contain the universal sequence used in the first step and the Illumina grafting primers (Forward Primer: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; Reverse Primer: 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3′). .. For sequencing data analysis, a Phred score of at least 23 for each base in the 36-base tags was applied to select high quality tags for mutation analysis.

Isolation:

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: Genomic DNA was isolated from cells in 1 ml of overnight culture using quick-gDNA miniprep kit (Zymo Research). .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing.

Size-exclusion Chromatography:

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences. .. Because of low PCR efficiency, the first-round PCRs for WT1 and CEBPA were carried out as follows: For WT1 , PCR was performed over 40 cycles using Taq DNA polymerase (Qiagen) under a 3-step thermal cycling of 94°C 30 sec-55°C 30 sec-72°C 30 sec; for CEPBA ; v1.0, read depth > 100).

Purification:

Article Title: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs
Article Snippet: PCR products were purified on a 8% native polyacrylamide gel run at 200 V for 30 min. Amplicons of 140–170 nt were excised from the gel and recover by ethanol precipitation. .. PCR primers contained the Illumina P5 and P3 sequences together with degenerated barcodes for PCR duplicates removal.

Article Title: Heteroplasmic mitochondrial DNA mutations in normal and tumor cells
Article Snippet: In the second step PCR, one pair of PCR primers were designed to contain the universal sequence used in the first step and the Illumina grafting primers (Forward Primer: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; Reverse Primer: 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3′). .. After 15 cycles of PCR using Phusion Flash master mix (NEB), the product was purified with a Qiaquick purification column (cat# 28104, Qiagen) and quantified by absortion at 600 nm.

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
Article Snippet: .. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR. .. All libraries were quantified using a Qubit Fluorometer (Invitrogen) and assessed on a 2% agarose gel.

Polymerase Chain Reaction:

Article Title: Transcriptome Sequencing Identifies PCAT-1, a Novel lincRNA Implicated in Prostate Cancer Progression
Article Snippet: .. RNA-Seq library preparation 2μg total RNA was selected for polyA+ RNA using Sera-Mag oligo(dT) beads (Thermo Scientific), and paired-end next-generation sequencing libraries were prepared as previously described using Illumina-supplied universal adaptor oligos and PCR primers (Illumina). .. Samples were sequenced in a single lane on an Illumina Genome Analyzer I or Genome Analyzer II flowcell using previously described protocols.

Article Title: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs
Article Snippet: .. PCR primers contained the Illumina P5 and P3 sequences together with degenerated barcodes for PCR duplicates removal. .. The iCLIP libraries were sequenced on a NextSeq 500 using 75-nt single-read sequencing runs.

Article Title: Heteroplasmic mitochondrial DNA mutations in normal and tumor cells
Article Snippet: .. In the second step PCR, one pair of PCR primers were designed to contain the universal sequence used in the first step and the Illumina grafting primers (Forward Primer: 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; Reverse Primer: 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3′). .. The PCR was conducted in a single PCR tube using this pair of primers and the diluted PCR product mixture from first step as template.

Article Title: Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis
Article Snippet: .. In all cases, purified adapter ligated DNA templates were then amplified through PCR enrichment, using PCR primers (Illumina), a dNTP mix, Phusion Polymerase (NEB) and 16 cycles of PCR. .. All libraries were quantified using a Qubit Fluorometer (Invitrogen) and assessed on a 2% agarose gel.

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing
Article Snippet: .. The PCR primers also encompassed p5 and p7 sequences that allow sequence capture and sequencing-by-synthesis on the Illumina GAIIx platform. .. To enable sufficient representation of each shRNA in the screening pool, multiple PCR reactions were performed in parallel to generate the sequencing library from each shRNA pool.

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites
Article Snippet: MGS-PCR utilizes exponential PCR coupled with nested PCR to rapidly identify RIS and allows for analysis of the number of different span or shear lengths to evaluate clonality. .. For adaptation, modified nested PCR primers including the Illumina universal adapters, indices, and stems for MiSeq paired-end sequencing were prepared ( ).

Article Title: Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila
Article Snippet: .. The PCR primers contained overhangs so that Illumina clustering, indexing, and sequencing oligonucleotides could be added in a subsequent nested PCR. .. The same primers designed for ratchet point lariat amplification were also used in different combinations to attempt to amplify the branchpoint lariats that would be generated by skipping one or more ratchet points.

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: .. The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences. .. PCR products were Illumina sequenced.

Article Title: The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms
Article Snippet: .. However, we have previously used this DNA extraction procedure in combination with the PCR primers targeting the V3 region of the 16S rRNA gene (absent the Illumina adapters and barcodes) to characterize a nitrifying enrichment culture in which Nitrosomonas spp. comprised as much as 25 to 70% of the bacterial community , again suggesting that analytical biases against the AOB of the order Nitrosomonadales are unlikely. .. A second (but much weaker) line of evidence supporting the claim that novel ammonia-oxidizing organisms are present in the BAC filters is the exceptionally high ratio of NOB to AOB (NOB/AOB ratio of 225) in the Illumina MiSeq profiles.

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing. ..

Article Title: Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy
Article Snippet: .. PCR primers included Illumina TruSeq sequencing primer sequences on the 5′ ends ( ). .. A second round of PCR was then used to add Illumina flowcell binding sequences and experiment-specific indexes 5′ of the primer sequence.

Article Title: Genome-Wide Profiling of Cap-Independent Translation Enhancing Elements in the Human Genome
Article Snippet: .. To detect and trim the PCR primers at both ends of each Illumina read, we used the “cutadapt” program ( http://code.google.com/p/cutadapt/ ) allowing a maximum of 2 mismatches. ..

Article Title: Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir
Article Snippet: .. Summary of pcr primers used in clone library and Illumina sequencing preparation. ..

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: .. PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences. .. Because of low PCR efficiency, the first-round PCRs for WT1 and CEBPA were carried out as follows: For WT1 , PCR was performed over 40 cycles using Taq DNA polymerase (Qiagen) under a 3-step thermal cycling of 94°C 30 sec-55°C 30 sec-72°C 30 sec; for CEPBA ; v1.0, read depth > 100).

CRISPR:

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: If a new spacer was integrated into the CRISPR array, the resulting PCR product was longer due to the additional repeat and spacer sequence. .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing.

Nested PCR:

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites
Article Snippet: .. For adaptation, modified nested PCR primers including the Illumina universal adapters, indices, and stems for MiSeq paired-end sequencing were prepared ( ). .. We performed Illumina MiSeq paired-end sequencing on MGS-PCR samples that had expanded clones, or were oligoclonal, or highly polyclonal: (1) lentiviral vector-mutagenized orthotopic primary prostate tumors that had expanded clones, (2) gammaretroviral vector-mutagenized CD105+ , Sca-1+ -enriched mouse bone marrow that was expanded in vitro and was oligoclonal, and (3) foamy viral vector-mutagenized human CD34+ cells that were highly polyclonal.

Article Title: Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila
Article Snippet: .. The PCR primers contained overhangs so that Illumina clustering, indexing, and sequencing oligonucleotides could be added in a subsequent nested PCR. .. The same primers designed for ratchet point lariat amplification were also used in different combinations to attempt to amplify the branchpoint lariats that would be generated by skipping one or more ratchet points.

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing. .. Lastly, a nested PCR was done to add additional sequences corresponding to Illumina adapters and barcodes.

Plasmid Preparation:

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites
Article Snippet: Here we set out to adapt the high-throughput MGS-PCR protocol published by Beard et al. for vector RIS analysis with the Illumina MiSeq paired-end sequencing platform ( ). .. For adaptation, modified nested PCR primers including the Illumina universal adapters, indices, and stems for MiSeq paired-end sequencing were prepared ( ).

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: .. The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences. .. PCR products were Illumina sequenced.

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: Spacer acquisition assay and high throughput sequencing Twenty colonies of P. furiosus strains transformed with plasmid were inoculated in 5 ml defined medium containing 20 μM uracil. .. PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing.

Multiplex Assay:

Article Title: Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy
Article Snippet: PCR primers included Illumina TruSeq sequencing primer sequences on the 5′ ends ( ). .. The resulting products were pooled and multiplex sequenced with 250 bp paired-end reads on an Illumina MiSeq instrument.

shRNA:

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing
Article Snippet: PCR primers complementary to constant regions found in all shRNA constructs (Figure ) were used to amplify the shRNA target sequence that is specific to each individual shRNA construct. .. The PCR primers also encompassed p5 and p7 sequences that allow sequence capture and sequencing-by-synthesis on the Illumina GAIIx platform.

Agarose Gel Electrophoresis:

Article Title: Genome-wide Identification of Zero Nucleotide Recursive Splicing in Drosophila
Article Snippet: The PCR primers contained overhangs so that Illumina clustering, indexing, and sequencing oligonucleotides could be added in a subsequent nested PCR. .. The primer pairs described above were used for the first-round PCR, after which products were visualized on an agarose gel.

Ethanol Precipitation:

Article Title: RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs
Article Snippet: PCR products were purified on a 8% native polyacrylamide gel run at 200 V for 30 min. Amplicons of 140–170 nt were excised from the gel and recover by ethanol precipitation. .. PCR primers contained the Illumina P5 and P3 sequences together with degenerated barcodes for PCR duplicates removal.

Next-Generation Sequencing:

Article Title: Transcriptome Sequencing Identifies PCAT-1, a Novel lincRNA Implicated in Prostate Cancer Progression
Article Snippet: .. RNA-Seq library preparation 2μg total RNA was selected for polyA+ RNA using Sera-Mag oligo(dT) beads (Thermo Scientific), and paired-end next-generation sequencing libraries were prepared as previously described using Illumina-supplied universal adaptor oligos and PCR primers (Illumina). .. Samples were sequenced in a single lane on an Illumina Genome Analyzer I or Genome Analyzer II flowcell using previously described protocols.

Article Title: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing
Article Snippet: Barcode recovery We used next generation sequencing to identify the frequency of each shRNA construct in screen cell populations. .. The PCR primers also encompassed p5 and p7 sequences that allow sequence capture and sequencing-by-synthesis on the Illumina GAIIx platform.

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites
Article Snippet: The MGS-PCR protocol can be adapted to other NGS platforms as older platforms are discontinued. .. For adaptation, modified nested PCR primers including the Illumina universal adapters, indices, and stems for MiSeq paired-end sequencing were prepared ( ).

Article Title: Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus
Article Snippet: Paragraph title: Spacer acquisition assay and high throughput sequencing ... PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing.

Multiple Annealing and Looping–Based Amplification Cycles:

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: After single-cell genome amplification with a MALBAC Single Cell WGA Kit (Yikon Genomics), target regions of genes of interest were PCR-amplified with primers including well indexes by PCR. .. PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences.

BAC Assay:

Article Title: pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping
Article Snippet: Paragraph title: BAC end sequencing and local assembly ... The 3΄ ends of the PCR primers are complementary to the vector backbone while the 5΄ ends contain Illumina adapter sequences.

Article Title: The Bacterial Communities of Full-Scale Biologically Active, Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms
Article Snippet: Also, similar results were obtained for other samples collected from both pilot-scale (data not shown) and full-scale BAC filters at SPRWS targeting the V3 region of the 16S rRNA gene (see Data Sets S6 and S7 in the supplemental material), suggesting that PCR bias against AOB of the order Nitrosomonadales was unlikely because it would be highly improbable for two PCR protocols targeting different regions of the 16S rRNA gene to be similarly biased against the same phylogenetic group. .. However, we have previously used this DNA extraction procedure in combination with the PCR primers targeting the V3 region of the 16S rRNA gene (absent the Illumina adapters and barcodes) to characterize a nitrifying enrichment culture in which Nitrosomonas spp. comprised as much as 25 to 70% of the bacterial community , again suggesting that analytical biases against the AOB of the order Nitrosomonadales are unlikely.

High Throughput Screening Assay:

Article Title: Modified Genomic Sequencing PCR Using the MiSeq Platform to Identify Retroviral Integration Sites
Article Snippet: Here we set out to adapt the high-throughput MGS-PCR protocol published by Beard et al. for vector RIS analysis with the Illumina MiSeq paired-end sequencing platform ( ). .. For adaptation, modified nested PCR primers including the Illumina universal adapters, indices, and stems for MiSeq paired-end sequencing were prepared ( ).

FACS:

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: Single-cell variation analysis was carried out for single cells sorted on a BD FACS Aria into 96-well plates. .. PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences.

Variant Assay:

Article Title: Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways
Article Snippet: PCR conditions were as in the first-round PCR, except that PCR primers were replaced with those for attaching Illumina anchoring sequences. .. Variant detection and frequency calculation were done with mpileup in SAMtools.

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