( s clearly depicted in Figure 3. The thr...)Real-Time PCR: General Considerations, Rev A

s clearly depicted in Figure 3. The threshold cycle was shifted by 4.7 cycles at 10⁴ copies and by 3.6 cycles at 10⁷ copies when the MgCl₂ concentration was increased from 1.5 mM to 3.0 mM. This significant shift in CT was not observed in the 2.25 mM or 4 mM MgCl₂ samples.

PCR efficiency can be affected by numerous factors. In this technical note we have evaluated 2 of these factors secondary structure interference and free magnesium ion concentration. We have clearly demonstrated that optimization of PCR design and reaction conditions on this template has strong effects on the quality of real-time PCR assays.

Once the primer locations and the MgCl₂ concentrations have been evaluated, other reaction components, such as probe concentration and protocol temperatures, may need to be evaluated. For example, a 2-step or 3-step PCR thermal protocol may yield optimal amplification detection, depending on the detection strategy. Although this initial optimization may seem cumbersome, it will result in reaction conditions that are robust and reproducible.

* SYBR is a trademark of Molecular Probes, Inc. Practice of the patented polymerase chain reaction (PCR) process requires a license. The iCycler iQ system includes a licensed thermal cycler and may be used with PCR licenses available from PE Corporation. Its use with authorized reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Some applications may require licenses from other parties.

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