( abeled probe (see Table) and 196 l of d...)Identification of Nonspecific Products Using Melt-Curve Analysis on the iCycler iQ Detection System, Rev A

A series of consecutive 5-fold dilutions of the IL-1β plasmid in ddH₂O was made, creating five dilutions from 10⁴ to 16 copies of the plasmid. The master mix was dispensed in 294 l aliqouts to six separate tubes. Then a 6 l aliquot from each plasmid dilution was added to one of the tubes containing master mix, with the last tube receiving 6 l of ddH₂O as a no-template control.

Primers for the experiment in Figure 7 were redesigned to prevent primer-dimer formation. The following master mix was used to generate this standard curve using SYBR Green I as the detection agent:

1.425 ml Platinum PCR Super mix (Life Technologies)
8.55 l 100 M forward primer (see Table)
8.55 l 100 M reverse primer (see Table)
285 l SYBR Green I (1:10,000 dilution of Molecular Probes stock)
1.065 ml ddH₂O

A series of consecutive 10-fold dilutions of the IL-1β plasmid template DNA ranging from 10⁷ copies/l to 10² copies/l was generated. Master mix was dispensed in 343 l aliquots to seven separate tubes. Each of the tubes then received a 7 l aliquot of one of the DNA dilutions. ddH₂O (7 l) was added to the last tube as a no-template control.

Aliquots (50 l) of the reaction mixtures were transferred to the wells of a 96-well thin-wall PCR plate. The plate was sealed with optically clear sealing film and spun briefly. Each plate was subjected to a PCR amplification protocol of 95C denaturation for 3 min followed by 4045 cycles of 10 sec at 95C and 45 sec at 5560C (annealing and data collection
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