title: cDNA sequences of Jatropha curcas: pool of six seed development stages
design: Equal amounts of extracted RNA from six different seed developmental stages were pooled and used for the synthesis of the first-strand cDNA from one g mRNA. cDNA were digested by SfiI restriction enzyme, fragments over 800 bp were isolated from an LMP agarose gel was used for ligation into pDNR-lib Vector and transformed into NEB10b competent cells. One million clones were plated on LB-Cm agar plates, collected and stored in glycerol stocks at -70C. One-half of the cells were inoculated to a 300 ml Terrific Broth/Cm culture. Plasmid DNA was purified, inserts were cut using SfiI restriction endonuclease, isolated from agarose gel and ligated to high-molecular-weight DNA using an Sfi-linker. The library for the Roche 454 FLX sequencing was generated according to the manufacturers protocols. The concatenated inserts were sheared to fragments ranging from 400 to 900 bp. The two 454 A and B adaptors were ligated to the ends of the emulsion PCR and sequencing. The library was sequenced on one picotiter-plate of the GS FLX using the Roche/454 Titanium chemistry.
library_name: JCS_454_seed