Sequencing of 3'-end of transcripts of a not normalized grape berry library
The cDNA libraries were prepared by Eurofins MWG Operon, Ebersberg, Germany (www.eurofinsdna.com) in cooperation with Vertis Biotechnologie AG, Freising, Germany. In short: After enrichment of polyA  RNA, the first strand cDNA was synthesized using an oligo(dT)-adapter primer. After purification of the first strand cDNA, second strand cDNA synthesis was performed using a random (N)6-adapter primer. With an aliquot of the cDNA, normalization was carried aout by one cycle of denaturation and reassociation of the cDNA (cot curve). The reassociated double strand cDNA was separated from the remaining single strand cDNA (i.e. the normalized cDNA) by passing the mixture over a hydroxyl apatite column. After hydroxyl apatite chromatography, the single stranded cDNA was subjected to 8 PCR cycles. Fragments in the size range of 450-550 bp were eluted from preparative agarose gels. An aliquot of the size fractionated cDNA was analyzed on a 1.5% agarose gel.