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
EL492641EL492641EST
EL492642EL492642EST
EL492643EL492643EST
EL492644EL492644EST
EL492645EL492645EST
EL492646EL492646EST
EL492647EL492647EST
EL492648EL492648EST
EL492649EL492649EST
EL492650EL492650EST
EL492651EL492651EST
EL492652EL492652EST
EL492653EL492653EST
EL492654EL492654EST
EL492655EL492655EST
EL492656EL492656EST
EL492657EL492657EST
EL492658EL492658EST
EL492659EL492659EST
EL492660EL492660EST
EL492661EL492661EST
EL492662EL492662EST
EL492663EL492663EST
EL492664EL492664EST
EL492665EL492665EST

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