title: GFNChile_Peach_0.9K_v1.0
status: Public on Apr 30 2007
contact.city: Santiago
contact.first: Mauricio
contact.last: Gonzalez
contact.email: maugonzalez@inia.cl
contact.state: Region Metropolitana
contact.address: Santa Rosa 11610
contact.country: Chile
contact.institute: Instituto de Investigaciones Agropecuarias
organism: Prunus persica
manufacture_protocol: The source of the spotted cDNA was a cv. Loring ripe peach fruit (Prunus persica L. Batsch) cDNA library. The library was constructed in the pBluescript II SK(-) vector and acquired in Clemson University Genomics Institute. A total of 847 cDNAs were selected from this library based on the functional properties assigned to their encoded proteins using an in-house gene-ranking method. This analysis involved sequence homology search using tBLASTx of 3,843 non-redundant sequenced clones from the cDNA library against the Arabidopsis CDS database to generate a list of orthologs (e-value <1E-10, identity > 70% over a minimum of 50 aa) which were further analyzed by manual searches in the annotated database, gene ontology terms, and information obtained by literature. Sequence management was performed with database tools developed in our laboratory. Domain-based analyses used SMART and Interpro. Signal peptide prediction and subcellular localization used SignalP and TargetP v1.01 software. Selected EST clones were picked from freezer stocks and grown overnight in 96 well plates in 200 µl LB plus 50 mµ/mL of ampicillin. Overnight cultures were used directly as templates in PCR reactions containing 10X buffer, 0.25 mM each of dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq DNA polymerase and 0.2 µM of T3 and T7 forward/reverse primers. Aliquots (5 µl) of each reaction were loaded onto a 1.0% (w/v) agarose gel to determine product quality. 20 µL of PCR products were arrayed in 96-well plates and mixed with an equal volume of 50% of dimethyl sulfoxide. PCR products (70 ng) were single spotted onto nylon membranes (10 x 7 cm) Immobilon NY+ (Millipore) using an 8 pins print-head (Arraylt model SSP015) and the arraying robot Versarray Chip Writer Compact (BioRad). The membranes were treated and the cDNA was fixed to the membrane by UV-crosslinking using a Ultraviolet Crosslinker CL-1000 (UVP). In addition to the selected ESTs, the following controls were spotted onto membranes: (1) a fragment of the vector pBluescript II obtained by amplification with the T3 and T7 universal primers; (2) several aliquots of DMSO 50%; (3) PpExpansin (GeneBank: AB029083), a gene expressed in peach and (4) cDNA from a B. subtilis gene dap (obtained from ATCC; number 87486) used as spike mRNA to normalize between membranes.