Center for Biosystems Research, University of Maryland Biotechnology Institute
Manufacture protocol
The selected 3864 ESTs, representing approximately 2,200 unique genes, were ordered to form the array EST (AEST) library used for printing. Purified cDNAs were resuspended in 50 μm 3X SSC and arrayed using Affymetrix 417 Arrayer on poly-lysine-coated glass slides with an average spot diameter of 100 μm and a spot spacing of 300 μm. After printing, the spotted cDNA was cross-linked to the surface of the slides (at 65 mJ) by using a StrataLinker instrument and washed with a 1% SDS solution to minimize the background. Slides were subsequently placed in a blocking solution containing 0.2 M succinic anhydride and 0.05 M sodium borate prepared in 1-methyl-2-pyrrolidinone for 20 min. washed for 2 min in 95 C water, and rinsed five times in 95% ethanol. Slides were spin dried at 500 rpm for 5 min and stored for hybridizations.
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