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Amplification of Mouse cDNAs for Microarrays Using the Eppendorf ,,, MasterTaq Kit

After printing, the microarrays were allowed to dry completely at ambient conditions. The slides were pretreated with 2x SSPE, 0.2% SDS at 55C. The cDNA probe was lyophilized and redissolved in ~32 l Microarray Hybridization Buffer Version 2 (APBiotech, UK). The solution was added to the microarray and a coverslip was applied. Hybridization was allowed to proceed for 1418 hours at 42C. The microarray was washed with 1X SSC/0.2% SDS for 5 minutes at 45C followed by two 5-minute washes with 0.1X SSC/0.2% SDS at room temperature. The microarray was rinsed briefly with water, dried with nitrogen, and scanned using a Molecular Dynamics Generation III scanner.

Results and Discussions

For the analysis of gene expression in eukaryotes, expressed sequence tag (EST) data represent an invaluable resource for gene identification. EST's are single pass partial cDNA sequences. In general, cDNA clones are selected to represent as many unique transcripts as possible. One such method to group transcripts is the Unigene clustering of data sets which attempts to identify unique human transcripts within EST data (http://www.ncbi.nlm.nih.gov/UniGene). In amplifying EST collections for the production of microarrays, the yield of each PCR amplicon is a very important consideration. The optimal concentration of DNA for successful microarray experimentation is typically 200 g/ml. Hybridizations using non-covalent attachment chemistries (e.g. polylysine, aminosilane) generally work poorly with DNA spotted at concentrations lower than this. When amplifying thousands of clones, the main objective is to minimize the total number of reactions needed to generate sufficient material for printing. A high yield o
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