Bauer Laboratory, FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA
Manufacture protocol
Yeast microarrays were constructed using the amplifying to levels required for preparation of DNA microarrays by PCR (27) a set of clones containing 6218 verified ORFs of the yeast genome obtained from Research Genetics (Huntsville, AL). Some of the longer ORFs were amplified with the GIBCO/BRL Elongase Amplification Kit (Life Technologies, Rockville, MD), using 40 cycles of 1 min at 95 °C denaturation, 1 min at 55 °C annealing, and 10 min at 68 °C elongation. We obtained an amplified product confirmed by agarose gel electrophoresis for 98% of the ORFs. The amplified DNA was precipitated with isopropanol, washed with 70% EtOH, and resuspended in 25 μl Micro Spotting Solution (Telechem, Sunnyvale, CA). The DNA was spotted on CMT-GAPS amino-silane-coated glass slides (Corning), using a microarraying robot with a 16-pin head constructed from a design by Patrick O. Brown (http://cmgm.stanford.edu/pbrown/).