GST amplicon structure. (A) Schematic representation of the GST format, with the 40 extensions allocated to the 24 columns, c, and 16 rows, r, in a 384-multiwell plate. (B) Primary amplification of a GST (thick double line with large arrow) from genomic DNA (thin double line) with a pair of primers each containing a gene-specific portion (horizontally striped) and 5′-extension (gray). The large arrow indicates the orientation of the GST with respect to the direction of transcription. Elements are not drawn to scale. (C) Secondary amplification of a GST with PCR addition of Gateway attB1 and attB2 sites. (D) Structure of the GST entry clone after Gateway recombinational cloning of the amplicon in pDONR207. (E) Secondary amplification results illustrated for 48 GSTs (see Supplemental Table S2). (F) Tertiary PCR validation of BP reactions. GST insert size verification is carried out by amplification of the attL1-GST-attL2 cassette with the DNR5 and DNR3 primers and results in the synthesis of a DNA fragment carrying 167 and 487 bp beyond the original 5′- and 3′-extensions of the primary GST, respectively (see Supplemental Table S2).
