N. Louise Glass's lab in Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA.
Manufacture protocol
To construct a gene-specific microarray of N. crassa genome, we designed 70-mer oligonucleotide immobilized probes using ArrayOligoSelector, and the approximately 10,000 open reading frames (ORFs) derived mainly from the N. crassa databases at the Broad Institute and MIPS. ArrayOligoSelector selects a unique segment to identify each ORF considering the predicted sequences of all genes. The program avoids selecting internal self-annealing structures and internal repeat sequences, and preferentially chooses oligonucleotides within a narrow range of GC content, which are biased towards the 3' terminal region of each gene. Oligomers were synthesized (Illumina, San Diego, CA) for all ORFs. Analysis of oligomer placement in predicted genes indicated the desired strong 3' bias. Oligomers were re-suspended in 3x SSC to a final concentration of 20µM, and spotted. Eight 40mM ArrayControl Sense Oligo spots, which were complementary to the eight ArrayControl RNA spikes (Ambion, Austin, TX, USA), were also included. Amplified DNA may be precipitated in 96-well format with isopropanol, washed with 70% EtOH, and resuspended in a salt spotting solution such as 3X SSC. DNA is commonly spotted on polylysine-coated glass slides or γ-aminopolysilane (GAPS)–coated glass slides (Corning, Corning, NY), using a microarraying robot. GAPS slides are more expensive but have a better shelf life and tend to be less variable in quality than poly-lysine coated slides. 10,918 oligomers are on the chip. 10,526 oligos are based on MIPS or Broad Institute predicted ORFs. 8 oligos are Abmion ArrayControl Sense Oligo Spots (#1781). The remaining 384 oligos (ChIP_Chip) were mainly designed in the intergenic regions. 40uM oligos in 3x SSC were printed onto Corning UltraGAPS slides. Slides are not post-processed. Rehydration is optional. Oligos need to be cross-linked with 600mJ UV. Slides should be stable for a year at room temperature in desiccator. Slides may display unacceptable levels of green background and the way we know to remove is using the Pronto!™ Background Reduction Kit (Item #40029) from Corning. It is not 100% effective so we recommend prescanning after prehybridization and identifying spots that are 3x standard deviation above background for possible elimination. For fast, easy, successful fist microarrays, we recommend to use Corning/Promega kit: 40076 Pronto Plus Indirect System and RPN5661 Amersham Post-labeling Reactive Dye Pack, which are expensive but always give brilliant results.
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