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
EL492590EL492590EST
EL492591EL492591EST
EL492592EL492592EST
EL492593EL492593EST
EL492594EL492594EST
EL492595EL492595EST
EL492596EL492596EST
EL492597EL492597EST
EL492598EL492598EST
EL492599EL492599EST
EL492600EL492600EST
EL492601EL492601EST
EL492602EL492602EST
EL492603EL492603EST
EL492604EL492604EST
EL492605EL492605EST
EL492606EL492606EST
EL492607EL492607EST
EL492608EL492608EST
EL492609EL492609EST
EL492610EL492610EST
EL492611EL492611EST
EL492612EL492612EST
EL492613EL492613EST
EL492614EL492614EST

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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