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

Katrine Verdun, Richard Rouse, and Gary Hardiman
BIOGEM, Division of Biology, University of California San Diego, La Jolla, CA 92093-0349
e-mail: ghardiman@ucsd.edu
www.biogem.ucsd.edu

Introduction

Microarray technology, primarily advanced by Pat Brown and his colleagues at Stanford University, makes use of a robotic spotting device or "microarrayer" to spot DNA sequences onto "derivatized" glass slides (1). These arrays are then hybridized with cDNA probes and analyzed using methods conceptually similar to those described for Affymetrix gene chips (2). Since the cost of producing an individual array is relatively small, spotted arrays are highly versatile and can make use of a wide variety of clone sets. Microarrays thus enable individual investigators to perform large-scale analysis of model organisms or to customize arrays for special genome applications.

The cDNAs spotted on microarrays are typically PCR products, amplified by PCR from plasmid miniprep DNA of cDNA clones. Amplification directly from clones in culture has been recently reported (3). This is more advantageous in high-throughput applications in that it is more cost efficient, considerably less labor intensive, and minimizes sample cross-contamination. The recommended concentration for spotting on aminosilane and polylysine slides is 200400 fmol/l. Lower concentrations than this will result in poor signals, and higher concentrations are not recommended as target spots are seen to smear. Microarray hybridizations work poorly with weak PCR products. Consequently, PCR amplification is optimized to maximize DNA yield. In this report w
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