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===Some parameters to be considered=== *Starting material 10ng total RNA *Preheat thermal cycler to 45-60C depending on the temperature selected for cDNA synthesis (Protocol recommends 30min incubation at 55C as a starting point) *For high gc content templates use a 60C cDNA synthesis temperature *cDNA synthesis can be accomplished in a 15-30min incubation at 45-60C. For small targets and incubation time of 5min may be sufficient *Annealing temperature should be 10C below the melting temperature of the primers used *Extension time varies with the size of the amplicon (approximately 1 min per 1kb of amplicon) *For all targets up to 4.5kb, 2ul of superscript III RT/Platinum Taq Mix is sufficient
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