Figure 2

Rescue of the ABI3 coding region into a Gateway Entry vector. (A) Schematic diagram showing the location of primers and restriction enzyme sites used for analysis of the integrity of candidate clones of pWY148 encoding ABI3 in pJM1. Boxed region represents the ABI3 genomic coding region, and the line represents vector sequence. Arrows indicate location of diagnostic PCR primers and EcoRI sites used for evaluating the correctness of the assembled construct. (B) PCR-based screening of clones potentially encoding ABI3 in pJM1. The 5'-Test (primers Entry-L1 and ABI3-5'-CDS-Rescue-3'-Test) and 3'-Test (primers Entry-L2 and ABI3-3'-Term-Rescue-5'-Test) evaluates for the fusion of the vector with the 5' or 3' end of the ABI3 coding region. An amplicon of 0.4 kb or 0.5 kb is expected for correct vector-gene fusions at the 5' or 3' end, respectively. (C) Restriction enzyme analysis of candidate ABI3 clones in pJM1. Clones 1, 2, 3 and 6 from panel A were digested with EcoRI and exhibit the predicted restriction fragments of 0.8, 2.6 and 3.4 kb. M, DNA size marker (1 kb Plus ladder, Invitrogen) with representation from 0.1–1 kb (B) or 0.65–5 kb (C).