Microarray Production

For the initial set of arrays produced, 6361 intergenic regions were amplified using the Yeast Intergenic Region Primers (Research Genetics/Invitrogen). 50µL PCR reactions were performed in 96-well plates with each primer pair with the following conditions: 0.25 µM of each primer, 20 ng of yeast genomic DNA, 250 µM of each dNTP, 2 mM MgCl2, 1X PCR buffer (Perkin Elmer/Applied Biosystems), and 0.875 units of Taq DNA polymerase (Perkin Elmer/Applied Biosystems). PCR amplification was performed in MJ Research Thermocyclers beginning with 2 minute denaturation at 95°C, followed by 36 cycles of 30 seconds at 92°C, 45 seconds at 52°C, and 2 minutes at 72°C, with a final extension cycle of 7 minutes at 72°C. 1 µL of each PCR reaction mix was then reamplified in a 100 µL PCR reaction using universal primers (Life Technologies) with the same reagent concentrations and the following thermocycling conditions: 3 minutes at 94°C, followed by 25 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 1 minute at 72°C, with a final extension cycle of 7 minutes at 72°C. Each PCR product was verified by gel electrophoresis. The PCR products were then isopropanol precipitated, washed with 70% ethanol, dried overnight, and resuspended in 20 µL of 3X SSC. The resuspended DNA was transfered to 384 well plates and printed on GAPS-coated slides (Corning) using a Cartesian robot (Cartesian Technologies). The printed slides were rehydrated, snap-dried, and UV crosslinked in UV Stratalinker (Stratagene) set at 60 mJoules. The slides were then stored under vacuum for at least 2 days prior to hybridization. Prior to use, at least one slide from every batch printed was tested for quality.

Subsequent generations of arrays included approximately 400 additional intergenic regions as contained in an updated collection of Yeast Intergenic Region Primers (Research Genetics/Invitrogen). The protocol for the creation of these arrays was exactly the same as above with the exception that the DNA was printed using an OmniGrid 100 Microarrayer OmniGrid 100 Microarrayer.