A-MEXP-2325 - microarray_VD

Verticillium dahliae
cDNAs used in generating the arrays were selectedfrom the V. dahliae SXM and DMS libraries {Neumann, 2003 #66}. For amplification of cDNAs, clones were cherry-picked from the SXM and DMS libraries, grown in liquid culture, and DNA was isolated as follows. Selected clones from master library glycerol stocks were inoculated into Luria broth in 96-well growth blocks using a Multiprobe II EX Robotic Handling System (Packard Intrument Company, Downer�s Grove, USA) and cultured overnight at37oC. Plasmid DNA was isolated from the cultures using the Qiaprep 96 Turbo Miniprep system (Qiagen, Mississauga, Canada) automated using a Biomek 2000 Laboratory Automation Workstation (Beckman-Coulter, Mississauga, Canada). Individual cDNAs were amplified by PCR using T3 (5�-ATTAACCCTCACTAAAGGGA-3�) and T7 (5�-TAATACGACTCACTATAGGG-3�) primers. Amplification by PCR was performed in a thermocycler (GeneAmp PCR system 9700, Applied Biosystems, Streetsville, Canada) Each 25-ul PCR reaction contained approx. 50ng genomic DNA, 400nM primer, 200 uM each dNTP, 2.5mM MgCl2, 200 mM Tris-HCl (pH 8.4), 500 mM KCl, and 0.5 U Platinum Taq polymerase (Invitrogen, Canada Inc., Burlington, Canada). Denaturation of the template at 94oC for 2 min, was followed by 30 amplification cycles: 94oC for 45 sec, 65oC for 45 sec, 72oC for 1 min, with a final 5 min extension at 72o. To verify amplification success, products were separated by electrophoresis through 0.8% agarose gels, and visualized by staining with 0.5ug/ml ethidium bromide. Low-yielding amplification reactions were repeated using an annealing temperature of 63oC, and amplification reactions resulting in non-specific amplification were repeated at an annealing temperature of 68oC. Amplification products were purified by filtration through Sephadex G50 collumns (Amersham Biosciences, Baie d'Urf�, QC) in 96-well Polyfiltronics Unifilters (Fisher Scientific, Toronto, Canada) plates, desiccated using the Savant Speedvac Plus SC210 (Global Medical Instrumentation, Ramsey, USA) and re-suspended in 20ul of 50% dimethyl sulfoxide for a final cDNA concentration of approx 200ng/ul. Arrays were contructed by printing amplified cDNA targets on glass FMB cDNA slides (Full Moon BioSystmes, Inc., Sunnyvale, CA) using the VP 470 Manual Glass side Indexing unit and VP 478 8 pin Glass Slide Replicator according to manufacturer�s protocol ( V&P Scientific, Inc. San Diego, CA). Each array consisted of 768 spots in an 18 mm x 36 mm grid. Following spotting, slides were air-dried and UV cross-linked at 400mJ.
Dongquan Chen (dongquan@uab.edu)
Experiment E-MTAB-1793