title: SUCAST Sugarcane 2208 v1.0
status: Public on May 20 2007
contact.city: São Paulo
contact.first: Milton
contact.middle: Yutaka
contact.last: Nishiyama Jr
contact.email: yutakajr@gmail.com
contact.state: São Paulo
contact.address: Av. Professor Lineu Prestes, 748
contact.department: Biochemistry
contact.country: Brazil
contact.web_link: http://sucest-fun.org
contact.institute: Universidade de São Paulo - USP
organism: Saccharum officinarum
description: Six 384-well plates representing 1830 unique SUCAST SAS were rearrayed for spotting
manufacture_protocol: Sugarcane cDNA plasmid clones obtained from the SUCEST collection were re-arranged and amplified in 100ul PCR reactions (40 cycles, annealing at 51°C), directly from bacterial clones in culture, using T7 and SP6 primers. PCR products were purified by filtration using 96 well filter plates (Millipore Multiscreen MAFBN0B50). Samples were visualized on 1% agarose gels to inspect PCR-amplification quality and quantity. Purified PCR products (in 10mM Tris-HCl solution at pH 8.0) were mixed with an equal volume of DMSO in 384 well V-bottom plates. Microarrays were constructed by arraying cDNA fragments on DMSO optimized, metal coated glass slides (type 7, Amersham Biosciences) using the Generation III Microarray Spotter (Molecular Dynamics/Amersham Pharmacia Biotech). Each cDNA fragment was spotted on the slides at least twice (i.e., technical replicates). Following printing, the slides were allowed to dry and the spotted DNA was bound to the slides by UV-cross linking (50mJ).