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The iCycler iQ Detection System for TaqMan Assays, Rev B

called the threshold cycle, and there is a linear relationship between the log of the starting amount of a template and its threshold cycle during real-time PCR. Given known starting amounts of the target nucleic acid, a standard curve can be constructed by plotting the log of starting amount versus the threshold cycle. This standard curve can then be used to determine the starting amount for each unknown template based on its threshold cycle.

Performance of a real-time PCR instrument is usually characterized by the sensitivity, uniformity and dynamic range of linear response to a variety of input sample concentrations. The iCycler iQ system demonstrates excellent performance characteristics in these areas. In this application note we illustrate the performance of the system when used with TaqMan probes.


Methods
Uniformity
The following master mix was prepared:
4.55 ml Platinum PCR supermix (Life Technologies)
15 l 100 M forward primer (5-TGCGTGACATTAAGGAGAAG)
15 l 100 M reverse primer (5-GCTCGTAGCTCTTCTCCA)
5.6 l 180 M probe (5-FAM-CACGGCTGCTTCCAGCTCCTC-TAMRA)
100 l pEFGP-Actin (-actin) plasmid, diluted to 105 copies/l (Clontech)
314 l dd H2O

The master mix was thoroughly vortexed and then 50 l aliquots were transferred to the wells of a 96-well thin-wall PCR plate. The plate was covered with a piece of optically clear sealing film and spun briefly to bring all reagents to the bottom of the wells. PCR conditions were 3 min at 95C, followed by 40 cycles of 15 sec at 95C and 30 sec at 60C. Fluorescent data were collected at the 60C step. In the plate setup file, all wells were d
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