title: Eucalyptus Array #1
status: Public on May 19 2011
contact.city: Canberra
contact.first: Andrew
contact.last: Spriggs
contact.email: andrew.spriggs@csiro.au
contact.state: ACT
contact.address:  GPO Box 1600
contact.department: Bioinformatics Group
contact.country: Australia
contact.institute: CSIRO Plant Industry
contact.phone: 612-6246-5193
organism: Eucalyptus nitens
description: Random Eucalyptus cDNA
manufacture_protocol: A cDNA library of genes expressed in differentiating xylem tissue from a vertical stem of E. grandis was constructed using the Stratagene lambda ZAP®II pre-digested vector kit (Stratagene, La Jolla, CA) with poly (A)+mRNA isolated by methods described previously (Southerton et al., 1998). The primary library was amplified before pBluescript phagemids were mass excised using the ExAssit/SOLR system (Stratagene, La Jolla, CA). Randomly picked phagemid colonies were cultured in a 96-well plate and the inserts amplified by PCR directly using forward, 5'-GTAAAACGACGGCCAGTG- 3' and reverse, 5'-GGAAACAGCTATGACCATG- 3' primers. The 100 ?l PCR reaction mixture contained 10 ?l of 10X PCR (-Mg2+) buffer, 2 ?l of 10 mM dNTP mixture, 5 ?l of primer mix (10 ?M each), 77.5 ?l of MilliQ water, 0.5 ?l of Taq DNA polymerase (5U ?l-1, LifeTechnologies, Rockville, MD) and 2 ?l supernatant 1 from overnight culture. Reactions were amplified on a 9600 GeneAmp PCR System (Perkin-Elmer, Foster City,  CA) using the following PCR amplification programme: 3 min denaturation at 95 °C, 34 cycles of 30 s at 94°C, 30 s at 45°C and 2.5 min at 72°C, followed by a 7 min extension at 72°C. The PCR products were purified using the TIGR's filtration protocol (Hegde et al., 2000). After washing twice with 100 ?l of MilliQ water, 16 ?l of 3 X SSC was added to each well and shaken vigorously for 10 minutes on a shaker to resuspend the DNA. Approximately 4,900 cDNAs were printed onto Superaldehyde glass slides (TeleChem, Sunnydale, CA) using a Bio-Rad (Hercules, CA) VersArray Chipwriter Pro with SMP 3 stealth microspotting quill pins (TeleChem, Sunnydale, CA). Each cDNA was spotted twice by different pins on a different area of the same slide. Glass slides were processed according to the TeleChem protocols. Cy3-and Cy5-labeled (Amersham Pharmacia) cDNA probes were generated using the two-step labelling method described by Schenk et al. (2000). Application of the probe to microarray slides, hybridization, and subsequent washes of the slides were performed according to Schenk et al. (2000). For each tree, slides were hybridized with probes synthesized from vertical xylem and one or other of upper or lower branch xylem.