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The iCycler iQ Detection System for TaqMan Assays, Rev B

h) was partially digested by BamHI at 37C for 2 hr, then heated to 100C for 5 min before being plunged into an ice-water bath. A 2-fold dilution series was prepared from the genomic DNA, ranging from 100 ng/l to 781 pg/l. For each tube of the dilution series, 8.25 l was pipetted into one of the 8 skirted tubes and vortexed. Finally, for each of the tubes, eight 50 l samples were transferred into one column of a 96-well thin-wall PCR plate. The plate was prepared for PCR as above. PCR conditions were 3 min at 95C followed by 40 cycles of 10 sec at 95C and 30 sec at 60C. Data were collected during the 60C annealing/extension step. In the plate setup file, 4 wells of each replicate group within a column were defined as standards and the other 4 as unknowns.


Results
Uniformity
When the background-corrected data were brought down to the PCR baseline, the 96 wells had a mean threshold cycle of 23.4 with a standard deviation of 0.6 cycles and a coefficient of variation (CV) of 2.6%. However, these data could be optimized by first extending the baseline cycles to include cycles 224; this changes the mean threshold cycle to 25.5 with a standard deviation of 0.16 cycles (0.63% CV). Increasing the data window to include the last 75% of the data collected during the annealing/extension step, and changing the threshold to 32.5 relative fluorescence units (RFU) or 15 times the mean standard deviation during the baseline cycles further improved the data, so that the final mean threshold cycle was 25.9 with a standard deviation of 0.12 cycles with 0.48% CV (Figure 1).

Linearity
When the background-corrected data were brought down to the PC
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