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

e describe the use of the Eppendorf MasterTaq Kit in the preparation of targets for a mouse ~8 K microarray.

Materials and Methods

PCR amplification
Selected clones were inoculated into 96-well Falcon U-bottom plate (Nunc) containing LB/Ampicillin (50 mg/ml) and were placed in a shaking incubator (200 rpm) overnight at 37C. PCR reactions (50 l) were assembled using a Qiagen BioRobot 3000 and consisted of 10 M dNTP mix, 20 M primer mix (forward primer 5'-CTG CAA GGC GAT TAA GTT GGG TAA C-3' and reverse primer 5'-GTG AGC GGA TAA CAA TTT CAC ACA GGA AAC AGC-3') and 1 l of overnight culture. Amplifications were carried out using 7.5 Units of polymerase in the recommended buffer, in accordance with the instructions provided by the manufacturer. Thermocycling was performed on an MJ Research Tetrad. Cycling conditions were as follows: an initial five-minute denaturation at 95C to lyse the cells and release the plasmid DNA, 35 cycles (94C for 1 minute, 58C for 1 minute, 7 2C for 4 minutes), followed by 10 minutes at 72C. Conditions recommended by manufacturers of Hot Start polymerases typically include a 15-minute rather than a 5-minute initial denaturation step. One-fifth of the PCR amplification was analyzed on a 0.8% agarose gel containing ethidium bromide and was visualized with an AlphaImager 2200 Documentation and Analysis system.

The mouse GEM 1 Clone List (build 35) containing 8,734 clones was purchased from Incyte Pharmaceuticals (Palo Alto, CA). For amplification of this mouse Unigene set, reactions were carried out in a 96-well plate format using 3 l of overnight culture and either 7.5 or 3.75 U MasterTaq respectively, in a final volume of 150 l. Succe
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