title: LBEG_Peach_1.5k_v1.0
status: Public on Feb 23 2011
contact.city: Santiago
contact.first: Leonardo
contact.middle: Ignacio
contact.last: Pavez
contact.state: Santiago
contact.address: Av Manuel Montt #948
contact.country: Chile
contact.institute: Universidad de Las Américas
organism: Prunus persica
manufacture_protocol: The source of the spotted cDNA was a cv. Loring and cv. O´henry ripe peach fruit (Prunus persica L. Batsch) cDNA library. A total of 1463 cDNAs were selected from this library based on the functional properties assigned to their encoded proteins using an in-house gene-ranking method.  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) cDNA from a B. subtilis gene dap (obtained from ATCC; number 87486) used as spike mRNA to normalize between membranes.