Journal: PeerJ

Article Title: Quantitating primer-template interactions using deconstructed PCR

doi: 10.7717/peerj.17787

Figure Lengend Snippet: CS1, common sequence 1 linker sequence. CS2, common sequence 2 linker sequence. BC, barcode. F, Forward primer. R, Reverse primer, P5/P7, Illumina primers, PE1/PE2, Fluidigm Access Array Barcode Library Illumina adapters. In stage 1 (linear copying only), individual samples are cycled for four cycles with locus-specific primers and Fluidigm barcoded primers. Subsequently, all reactions are pooled and purified together, and then amplified with Illumina P5 and P7 primers in stage 2 (exponential amplification with primers targeting linker sequences). During stage 1, linear copying of templates leads to products which contain Illumina sequencing adapters, sample-specific barcodes, and the locus-specific region of interest. Only fragments with Illumina adapters and barcodes are exponentially amplified in stage 2. Locus-specific primer sequences can be modified as needed.

Article Snippet: A master mix for the entire plate was made using the MT master mix, and each well received a separate primer pair with a unique Access Array Barcode Library for Illumina primer (described above).

Techniques: Sequencing, Purification, Amplification, Illumina Sequencing, Modification

Journal: PeerJ

Article Title: Quantitating primer-template interactions using deconstructed PCR

doi: 10.7717/peerj.17787

Figure Lengend Snippet: CS1, common sequence 1 linker sequence. CS2, common sequence 2 linker sequence. BC, barcode. F, Forward primer. R, Reverse primer, P5/P7, Illumina primers, PE1/PE2, Fluidigm Access Array Barcode Library Illumina adapters. In stage 1 (linear copying only), individual samples are cycled for four cycles with locus-specific primers and Fluidigm barcoded primers. Subsequently, all reactions are pooled and purified together, and then amplified with Illumina P5 and P7 primers in stage 2 (exponential amplification with primers targeting linker sequences). During stage 1, linear copying of templates leads to products which contain Illumina sequencing adapters, sample-specific barcodes, and the locus-specific region of interest. Only fragments with Illumina adapters and barcodes are exponentially amplified in stage 2. Locus-specific primer sequences can be modified as needed.

Article Snippet: For barcoding reactions, Access Array Barcode Library for Illumina primers were purchased (Fluidigm, South San Francisco, CA, USA).

Techniques: Sequencing, Purification, Amplification, Modification