454 sequencing of Phaseolus coccineus Embryo-Proper Region transcript fragment library
Embryo-proper regions were micro-dissected from globular-stage embryos six days after pollination from greenhouse-grown plants [Weterings et al., Plant Cell 13, 2409-2425 (2001)]. Double-stranded cDNA was synthesized from embryo-proper mRNA using the SMART cDNA Library Construction Kit according to the manufacturer (Clontech). The embryo-proper cDNA fragments were directionally ligated into the SfiI restriction site of the lambda TriplEx2 vector (Clontech), and the recombinant cDNAs were transformed into E. coli XL1-Blue cells (Clontech). Embryo-proper cDNA plasmids used for directional sequencing were obtained by in vivo excision from the lambda TriplEx2 recombinants in E. coli BM25.8 cells (Clontech).