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
EL492691EL492691EST
EL492692EL492692EST
EL492693EL492693EST
EL492694EL492694EST
EL492695EL492695EST
EL492696EL492696EST
EL492702EL492702EST
EL492703EL492703EST
EL492704EL492704EST
EL492705EL492705EST
EL492706EL492706EST
EL492707EL492707EST
EL492708EL492708EST
EL492709EL492709EST
EL492710EL492710EST
EL492711EL492711EST
EL492713EL492713EST
EL492714EL492714EST
EL492715EL492715EST
EL492716EL492716EST
EL492717EL492717EST
EL492719EL492719EST
EL492720EL492720EST

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