title: Pine Secondary Xylem 4.0K SSH-cDNA array version 1.0
status: Public on May 06 2012
contact.city: Malaga
contact.first: Rocio
contact.last: Bautista
contact.email: rociobm@scbi.uma.es
contact.address: Edificio de Bioinnovación, C/ Severo Ochoa, 34
contact.country: Spain
contact.institute: Plataforma Andaluza de Bioinformatica, Universidad de Malaga
organism: Pinus pinaster
description: ONLY CLONES OF INTEREST HAVE BEEN SEQUENCED AND IDENTIFIED
manufacture_protocol: 1. Probes were PCR-amplified from four SSH libraries. SSH libraries were constructued with the PCR-select cDNA subtraction kit  (Clontech, Palo Alto, CA, USA). Two SSH libraries were produced using cDNAs from compression and opposite differentiating xylem by performing the subtraction procedure in both directions. Two additional SSH libraries were produced using cDNA from juvenile and mature differentiating xylem by the same procedure. Nested PCR primers 1 and 2R included in the kit were used for PCR amplification.
  2. PCR products were purified with 96-well multiscreen filter plates (Millipore Corp. Bedford, MA)
  3. Purified-cDNA probes in 50% DMSO were prepared to a concentration between 100 and 200 ng/µl in 384-well plates (Genetix)
  4. cDNA probe was printed in duplicate onto ULTRA gaps II coated slides (Corning, NY, USA) using a Qarray2 (Genetix Ltd, Queensway, UK) with a telechem printing head and 16 split pins (Biorobotics, Cambridge, UK) with a 4 x 4 configuration.
  5. After printing, the slides were dried at room temperature and the spotted cDNA were cross-linked to the slide surface by UV irradiation at 300 mJ/cm²