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Real-time PCR is best performed when all assays have 100% geometric efficiency. The proof of the system was demonstrated by a study of the efficiencies of 750 randomly selected TaqMan Gene Expression Assays (Amplification Efficiency of TaqMan Gene Expression Assays application note).
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The system integrates universal cycling conditions, chemistry and assay design. In the 1990s, Perkin Elmer Corporation scientists developed a universal system for real-time PCR that consistently produced a geometric efficiency of 100%. Therefore, assigning accurate efficiency values is important for producing accurate quantity results. For example, for a Ct of 20, the quantities resulting from 100% vs. In equation 1, efficiency (e) is a base in an exponential function, which means a change in the value of e can have a significant impact on the resulting quantity. That efficiency can be represented as 2 or 100%. At maximum, a target sequence can double each cycle, because DNA only has two strands. In the geometric phase, the efficiency is constant cycle-to-cycle.Įfficiency can be represented as a ratio or a percentage. A standard curve for the ideal target diluted concentration at each dilution point was calculated using the dilution factor of.
#Parallel dilution calculator serial#
Is parallel or serial dilution more accurate Which dilution is more accurate Serial dilutions made by Andrew are more accurate than their manually performed counterparts. PCR efficiency can be defined as the ratio of the number of target gene molecules at the end of a PCR cycle divided by the number of target molecules at the start of the same PCR cycle. A 1:10 dilution is also called a 10x dilution. For simplicity, the term “Ct” will be used in this document. Consequently, these geometric data points may have slightly different abbreviations, such as Ct, Crt or Cq, but they are all treated the same way in subsequent calculations. Quantitative data can be acquired from the geometric phase using a variety of methods, such as baseline-threshold or relative-threshold. Plateau phase data is not considered quantitative, unless special techniques are employed, such as those used for “digital PCR.” This final phase of PCR is called plateau. Changes in efficiency during linear phase become less and less consistent with increasing cycle number, so the data becomes less and less quantitative.Įventually, PCR efficiency may become so low that there is no appreciable target amplification. Linear phase is the second phase in PCR in which the efficiency declines cycle-to-cycle. At this point, geometric phase transitions to linear phase. For example, using highly concentrated target, such as plasmid, 20ml PCR reactions can produce a high quality standard curve spanning 9 logs or more.ĭuring PCR, the DNA target may accumulate to a high enough level that one of the PCR reagents will no longer be sufficient to support geometric amplification. The geometric phase is also remarkable in that efficiency does not change with original gene quantity over a wide range. The remarkable consistency of geometric amplification maintains the original quantitative relationships of the target gene across samples.