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ISHS Acta Horticulturae 839: I International Symposium on Biotechnology of Fruit Species: BIOTECHFRUIT2008

OPTIMIZATION OF THE PREPARATION OF DS CDNA FROM FLOWER BUDS OF APRICOT FOR THE CDNA-AFLP ANALYSIS

Authors:   J. Cechova, M. Baranek, M. Pidra
Keywords:   RNAlater®, SpectrumTM Plant Total RNA Kit, MINT cDNA Synthesis Kit
DOI:   10.17660/ActaHortic.2009.839.73
Abstract:
The aim of the work was to develop the preparation of ds cDNA from flower buds of four apricot varieties (‘Sundrop’, ‘Stark Early Orange’, ‘Vestar’ and ‘Betinka’) so that it would be as suitable as possible for the cDNA-AFLP analysis. The sampled tissues were preserved using a commercial agent RNAlater® Soln. (Ambion). The RNA from the chosen samples was isolated using a commercial isolation kit supplied by Sigma (SpectrumTM Plant Total RNA Kit), which supports the isolation of sufficient amounts of high quality RNA from these highly complicated tissues. Transcription from the entire RNA onto the ds cDNA was achieved using the MINT cDNA synthesis kit from Evrogen. The cDNA-AFLP analysis was carried out according to an amended procedure by Bachem et al. (1998) and the samples obtained from selective amplification were analysed on genetic analyser ABI-PRISM 310. The results obtained by another protocol (include silica extraction of RNA, GenElute mRNA Miniprep Kit and others) were compared with new ones. On the base of this comparison, suitable commercial agent, kits and procedures for the preparation of ds cDNA of sufficient quality have been identified and are recommended for similar type of work.
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