454 GS FLX+ sequencing; Comparative 454 pyrosequencing of transcripts to provide a resource for the study of embryogenesis of coconut (Cocos nucifera L.)
Comparative 454 pyrosequencing of transcripts to provide a resource for the study of embryogenesis of coconut (Cocos nucifera L.)
Developing leaves (Leaf) were sampled in vitro germinated embryo culture plant (Weerakoon et al 2002). In vitro cultures were maintained at 28 degree centigrade under dark conditions in a culture room. Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, UK) using manufacturers protocol. cDNA libraries were constructed using SMART approach as described by Zhu et al. 2001 using the the oligonucleotides SMART Sfi1A : 5prime- AAG CAG TGG TAT CAA CGC AGA GTG GCC ATT ACG GCCrGrGrG-3', CDS-Sfi1B : AAG CAG TGG TAT CAA CGC AGA GTG GCC GAG GCG GCCd(T)20-3',  SMART PCR primer : 5'-AAG CAG TGG TAT CAA CGC AGA GT- 3'. The amplified cDNA PCR products were purified using QIAquick PCR Purification Kit (QIAGEN, CA) and a final  yield of 3 microgram was prepared for each tissue type.