Citrus sinensis flesh cDNA-AFLP Library

Overview
Library NameCitrus sinensis flesh cDNA-AFLP Library
Unique NameCitrus sinensis flesh cDNA-AFLP Library
OrganismCitrus sinensis (Sweet orange)
Typecdna_library
Vector: PGEM Teasy vector; Site_1: BstYI; Site_2: MseI; Fruits were harvested at three developmental stages. Poly-A mRNA was isolated from total RNA extracted from flesh according to the Trizol LS and Dynabeads M-280 Streptavidin protocols. A cDNA-AFLP technique of RNA fingerprinting was optimized allowing the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent polymerase chain reaction (PCR) amplification. This is based on the selective amplification of cDNA fragments obtained by digestion with one frequent and one rare cutter enzymes, followed by ligation of two double-stranded adapters of known sequences. AFLP-TP (Transcriptional Profiling) is an improved version of the cDNA-AFLP protocol, that allows the isolation one unique restriction fragment for each cDNA. The esacutter enzyme is BstYI (recognition site Pu/GATCPy) and the fourcutter is MseI (recognition site T/TAA). The difference in AFLP-TP lies in an additional step between the two digestions: as the cDNA synthesis is obtained with a biotinylated oligo-dT, this allows to collect the 3 end of the transcript after the first digestion, taking advantage of the high affinity of the biotin for the streptavidin anchored to magnetic beads (Dynal). After the electrophoretic run, a vertical scanner collected the fluorescence emissions derived from the excitation of the fluorescein molecules attached to the BstYI primers with a 465nm monochromatic beam. Single fragments were isolated either through DGGE (Denaturing Gradient Gel Electrophoresis) analysis or cloning into pGEM-T Easy vector (Promega). Samples that showed only one neat band were sequenced.
SNP Chip Base
Array NameCitrus sinensis flesh cDNA-AFLP Library
OrganismCitrus sinensis (Sweet orange)
Typecdna_library
Features
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Feature NameUnique NameType
EL492412EL492412EST
EL492413EL492413EST
EL492414EL492414EST
EL492415EL492415EST
EL492416EL492416EST
EL492417EL492417EST
EL492418EL492418EST
EL492419EL492419EST
EL492420EL492420EST
EL492421EL492421EST
EL492422EL492422EST
EL492423EL492423EST
EL492424EL492424EST
EL492425EL492425EST
EL492426EL492426EST
EL492427EL492427EST
EL492428EL492428EST
EL492429EL492429EST
EL492431EL492431EST
EL492432EL492432EST
EL492433EL492433EST
EL492434EL492434EST
EL492435EL492435EST
EL492436EL492436EST
EL492437EL492437EST

Pages

Properties
Property NameValue
Fruittissue type
Genbank library cultivarBiondo cadenera,Tarocco nucellare 57-1E-I,Moro nucellare 58-8D-I
Genbank library dev stagethree developmental stages during ripening period
Genbank library noteVector: PGEM Teasy vector; Site_1: BstYI; Site_2: MseI; Fruits were harvested at three developmental stages. Poly-A mRNA was isolated from total RNA extracted from flesh according to the Trizol LS and Dynabeads M-280 Streptavidin protocols. A cDNA-AFLP technique of RNA fingerprinting was optimized allowing the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent polymerase chain reaction (PCR) amplification. This is based on the selective amplification of cDNA fragments obtained by digestion with one frequent and one rare cutter enzymes, followed by ligation of two double-stranded adapters of known sequences. AFLP-TP (Transcriptional Profiling) is an improved version of the cDNA-AFLP protocol, that allows the isolation one unique restriction fragment for each cDNA. The esacutter enzyme is BstYI (recognition site Pu/GATCPy) and the fourcutter is MseI (recognition site T/TAA). The difference in AFLP-TP lies in an additional step between the two digestions: as the cDNA synthesis is obtained with a biotinylated oligo-dT, this allows to collect the 3 end of the transcript after the first digestion, taking advantage of the high affinity of the biotin for the streptavidin anchored to magnetic beads (Dynal). After the electrophoretic run, a vertical scanner collected the fluorescence emissions derived from the excitation of the fluorescein molecules attached to the BstYI primers with a 465nm monochromatic beam. Single fragments were isolated either through DGGE (Denaturing Gradient Gel Electrophoresis) analysis or cloning into pGEM-T Easy vector (Promega). Samples that showed only one neat band were sequenced.
Genbank library tissue typeflesh