a working group funded by the National Institute of Dental and Craniofacial Research.
Manufacture protocol
C. albicans ORFs were obtained from assembly 6 of the C. albicans genome sequence (http://www-sequence.stanford.edu/group/candida). ORFs encoding proteins greater than 150 aa and that started with an ATG codon were included in the array. ORFs were sorted into two categories: those that were unique by BLAST comparisons, and those that were likely to represent a group of closely related genes or a gene family. Primers to amplify each of the unique ORFs were designed using PRIMER3 software (http://frodo.wi.mit.edu/primer3/primer3_code. html). Primers for the gene family ORFs were designed by hand and checked using the PRIMER3 program. Primers were designed to have a Tm of approximately 60 uC and to amplify a region of approximately 300 bp. When possible, amplification products were targeted to the 39 end of the coding region. Two separate rounds of PCR were conducted with each primer pair and C. albicans SC5314 genomic DNA as the template. PCR products from the reactions were combined and checked for quality by agarose gel electrophoresis. PCR products were purified using the MultiScreen-PCR 96-well filtration system (Millipore), eluted in water, dried in a SpeedVac (ThermoLabs) and resuspended at approximately 0.1ug/ul in 3X SSC (0.45 M NaCl, 0.045 M sodium citrate). A total of 6392 DNA samples were spotted onto SuperAmine Substrate slides (Telechem) at Microarrays Inc. Controls included Arabidopsis thaliana spot report genes and mammalian DNA fragments (Stratagene), C. albicans genes of known expression profiles, and buffer blanks.
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