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

ame Avg. Total ng/Plate 4W-6Z 5541.5 7W-9Z 5367.9 10W-12Z 5174.5 13W-15Z 4848.7 16W-18Z 5187.3 19W-21Z 4169.5 22W-6YAmm. 4401.6 Avg Total ng in Mouse EST 4955.9 Table 1
Yield of DNA from amplification of the mouse Unigene set. Eight samples from 96-well plates containing purified DNA were assayed for DNA concentration. The plates assayed were numbered 4W, 4X, 4Y, 4Z, 5W, etc. through 22Z. There were five additional plates called 24W, 24X, 24Z, 23W, and 6Yamm respectively.

Quantitation of amplicon yield

All of the PCR products were purified on a BioRobot 3000 (QIAGEN, Santa Clarita, CA) using the QIAquick 96 PCR BioRobot Kit. Representative DNA samples from the entire set of amplifications were then quantitated using the Picogreen kit (Molecular Probes) to determine an estimate of the yield of purified PCR product. The results are shown in Table 1. The mean clone total yield was 4955.9 ng. The DNAs were resuspended at a concentration of ~200 g/ml in 25 l 50% DMSO-H2O. Many microarray spotters use 200 nl per print run and have a void volume of ~5 l. Assuming the spotter can print 36 arrays per run, this equates in theory to approximately 3,600 microarray slides. The working number of slides however is lower than this due to sample evaporation and other printing variables.

Screening of a mouse microarray with cDNA probes generated from mouse
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