Technical Support FAQ

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Technical Support FAQ


Question:

Many investigators optimize both the temperature and magnesium concentration for their primer/probe combinations in one-step RT-PCR. When switching to a one-step probe kit, does Bio-Rad ever recommend adjusting the magnesium concentration?


Answer:

The magnesium concentration has already been optimized for the quantitative one-step RT-PCR kits. The iScript one-step RT-PCR kit with SYBR® Green and the iScript one-step RT-PCR kit for probes contain distinct concentrations of magnesium. Increasing the concentration of magnesium in the iScript one-step RT-PCR kit with SYBR® Green will likely increase the frequency of primer-dimers and other nonspecific PCR artifacts. For the iScript one-step RT-PCR kit for probes, magnesium concentration is set to support probe hybridization/hydrolysis as well as duplex quantitative RT-PCR. Using higher concentrations in this kit may also increase the likelihood of developing primer-dimers.

Testing different annealing temperatures is the most effective first course of action for optimizing these kits. A three-step cycling program may be the most effective strategy. The gradient feature on the iCycler iQ real-time detection system is a tool that can be used to determine optimal annealing temperature.

Failure to get a signal, or a highly delayed C T , is usually indicative of failed first-strand synthesis. Primers that work in a two-step RT-PCR procedure may not be suitable for one-step RT-PCR. Hence, primers may need to be redesigned. It is important to have an antisense primer that primes first-strand synthesis. Sliding a primer several bases along the sequence can have a profound impact on the efficacy of one-step RT-PCR. RNA secondary structure could interfere with annealing, extension, or both using one of these primers. Hence, RNA folding algorithms may facilitate the design of an effective antisense primer.


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