Structured Review

Pacific Biosciences multiplex pcr primer guidelines
Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct <t>PCR</t> sequencing, DNA sequencing of cDNA clones, and/or <t>PacBio</t> ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.
Multiplex Pcr Primer Guidelines, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm"

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm

Journal: Scientific Reports

doi: 10.1038/s41598-018-31840-5

Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.
Figure Legend Snippet: Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

Techniques Used: Binding Assay, Polymerase Chain Reaction, Sequencing, DNA Sequencing, Clone Assay

Related Articles

Multiplex Assay:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: .. The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ). .. Prior to SMRTbellTM library preparation, ~5 kb PCR products were gel purified with QIAquick® Gel extraction kit (Qiagen, Germantown, MD) and quantified using QubitTM dsDNA High Sensitivity Assay Kit with the QubitTM 2.0 Fluorometer (ThermoFisher Scientific).

Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing
Article Snippet: .. Full‐Length CYP2D6 Sequencing Using Direct Barcoding Barcoded fusion primers for amplification of full‐length CYP2D6 were designed based on the PacBio multiplex PCR primer guidelines. .. Initial analysis of one individual was performed using two technical replicate libraries with unique barcodes, prepared and sequenced together on one PacBio SMRT cell.

Amplification:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: Nested amplification was carried out with barcode-tailed PCR primers, 163pgABCA2-5 and 166pgABCA2-3 (Supplementary Table ). .. The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ).

Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing
Article Snippet: .. Full‐Length CYP2D6 Sequencing Using Direct Barcoding Barcoded fusion primers for amplification of full‐length CYP2D6 were designed based on the PacBio multiplex PCR primer guidelines. .. Initial analysis of one individual was performed using two technical replicate libraries with unique barcodes, prepared and sequenced together on one PacBio SMRT cell.

Sensitive Assay:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ). .. Prior to SMRTbellTM library preparation, ~5 kb PCR products were gel purified with QIAquick® Gel extraction kit (Qiagen, Germantown, MD) and quantified using QubitTM dsDNA High Sensitivity Assay Kit with the QubitTM 2.0 Fluorometer (ThermoFisher Scientific).

Ligation:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ). .. Barcoded samples (n = 22) were pooled in equimolar concentrations and SMRTbellTM libraries were prepared with 0.5 µg of the pooled amplicons following the standard procedures for blunt ligation of hairpin adapters using SMRTbellTM Template Prep Kit 1.0 (Pacific Biosciences).

Purification:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ). .. Prior to SMRTbellTM library preparation, ~5 kb PCR products were gel purified with QIAquick® Gel extraction kit (Qiagen, Germantown, MD) and quantified using QubitTM dsDNA High Sensitivity Assay Kit with the QubitTM 2.0 Fluorometer (ThermoFisher Scientific).

Polymerase Chain Reaction:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: .. The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ). .. Prior to SMRTbellTM library preparation, ~5 kb PCR products were gel purified with QIAquick® Gel extraction kit (Qiagen, Germantown, MD) and quantified using QubitTM dsDNA High Sensitivity Assay Kit with the QubitTM 2.0 Fluorometer (ThermoFisher Scientific).

Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing
Article Snippet: .. Full‐Length CYP2D6 Sequencing Using Direct Barcoding Barcoded fusion primers for amplification of full‐length CYP2D6 were designed based on the PacBio multiplex PCR primer guidelines. .. Initial analysis of one individual was performed using two technical replicate libraries with unique barcodes, prepared and sequenced together on one PacBio SMRT cell.

DNA Sequencing:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ). .. DNA sequencing was performed on the PacBio® RSII sequencer using P6-P4 enzyme chemistry at the University of Arizona, Arizona Genomics Institute (Tucson, Arizona).

Sequencing:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: Paragraph title: Targeted PgABCA2 Sequencing and Bioinformatics Analysis ... The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ).

Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing
Article Snippet: .. Full‐Length CYP2D6 Sequencing Using Direct Barcoding Barcoded fusion primers for amplification of full‐length CYP2D6 were designed based on the PacBio multiplex PCR primer guidelines. .. Initial analysis of one individual was performed using two technical replicate libraries with unique barcodes, prepared and sequenced together on one PacBio SMRT cell.

Gel Extraction:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ). .. Prior to SMRTbellTM library preparation, ~5 kb PCR products were gel purified with QIAquick® Gel extraction kit (Qiagen, Germantown, MD) and quantified using QubitTM dsDNA High Sensitivity Assay Kit with the QubitTM 2.0 Fluorometer (ThermoFisher Scientific).

Nested PCR:

Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm
Article Snippet: Because of limited available tissue and low abundance of the PgABCA2 transcript, we used nested PCR to generate amplicon templates for library preparation. .. The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ).

Software:

Article Title: Flexible and Scalable Full‐Length CYP2D6 Long Amplicon PacBio Sequencing
Article Snippet: Full‐Length CYP2D6 Sequencing Using Direct Barcoding Barcoded fusion primers for amplification of full‐length CYP2D6 were designed based on the PacBio multiplex PCR primer guidelines. .. Full‐length CYP2D6 sequences resulted after barcode demultiplexing and processing the data with the Long‐Amplicon Analysis software.

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    Pacific Biosciences multiplex pcr primer guidelines
    Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct <t>PCR</t> sequencing, DNA sequencing of cDNA clones, and/or <t>PacBio</t> ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.
    Multiplex Pcr Primer Guidelines, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/multiplex pcr primer guidelines/product/Pacific Biosciences
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    multiplex pcr primer guidelines - by Bioz Stars, 2020-04
    93/100 stars
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    Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

    Journal: Scientific Reports

    Article Title: ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm

    doi: 10.1038/s41598-018-31840-5

    Figure Lengend Snippet: Mutations affecting PgABCA2 protein in Cry2Ab-resistant pink bollworm from Arizona and India. ( a ) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini (pink), two transmembrane domains (TMD1 and TMD2), each consisting of 6 transmembrane regions (TM; orange), three extracellular loops (ECL; green), two intracellular loops (ICL; blue), and two nucleotide-binding domains (NBD; purple). Mutations affecting transcripts of resistant pink bollworm: Circles show premature stop codons from India (red), Arizona (yellow), or both (red and yellow). Triangles show in-frame indels from India (red) or Arizona (yellow). Numbers indicate the affected amino acids. ( b ) Full-length and partial PgABCA2 cDNAs were obtained by direct PCR sequencing, DNA sequencing of cDNA clones, and/or PacBio ® DNA sequencing from susceptible (APHIS-S) and resistant, laboratory-selected (Bt4-R2) pink bollworm from Arizona, USA and India field-selected resistant populations (AM, CK, GAP, KT, and RK). The linear schematic (top) shows the predicted translated domain structure of the 5,187-bp full-length PgABCA2 coding sequence. The predicted protein includes amino- and carboxyl-termini (pink), transmembrane regions TM1-TM12 (orange), intracellular loops ICL1-ICL5 (blue), and extracellular loops ECL1-ECL6 (green). The domain structure connected to the exons that encode the respective domains is shown by dotted gray lines. Each predicted domain is numbered, with ECLs on top and ICLs numbered on bottom of protein schematic. Putative exons 1–31 are numbered, with grey exons indicating regions determined by direct PCR sequencing. Exons colored in light blue were further verified by sequencing cDNA clones (either by Sanger or PacBio ® sequencing). Red bars indicate disruption sites within the full-length coding sequence and the red triangles indicate the location of premature stop codons shown to scale based on the linear schematic of the translated domain structure. Unique cDNA variants are indicated as a, b, c, etc.

    Article Snippet: The symmetric barcode-tailed PCR primers were designed based on the PacBio® multiplex PCR primer guidelines ( http://www.2einteractive.com/pacbio/Shared-Protocol-PacBio-Barcodes-for-SMRT-Sequencing.pdf ).

    Techniques: Binding Assay, Polymerase Chain Reaction, Sequencing, DNA Sequencing, Clone Assay