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The iCycler iQ Detection System for TaqMan Assays, Rev B

efined as unknowns.

Linearity
The following master mix was prepared:
450 l 10x Platinum PCR buffer (Life Technologies)
315 l 50 mM MgCl2
45 l 100 mM dNTP
13.5 l 100 M forward primer
13.5 l 100 M reverse primer
18 l Platinum Taq polymerase (Life Technologies)
5.0 l 180 M probe
3.55 ml dd H2O

This master mix was vortexed thoroughly and then 441 l of master mix was pipetted into 9 different 2.0 ml skirted tubes.

A stock of plasmid pEFGP-Actin encoding β-actin (Clontech) was quantitated using PicoGreendye (Molecular Probes) binding with the VersaFluor fluorometer (Bio-Rad), then diluted to 109 copies/l. A 10-fold dilution series was prepared from 109 to 10 copies/l, and then 9 l of each dilution was added to one of the skirted tubes containing master mix. Finally, eight 50 l aliquots of each mix were pipetted into wells of a single 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 50 cycles of 10 sec at 95C and 30 sec at 60C. Fluorescent data were collected during the 60C step. In the plate setup file, half the replicates from each set in the series were identified as standards and the other half as unknowns.

Sensitivity
The following master mix was prepared:
3.18 ml 1.1x Platinum PCR supermix
10.5 l 100 M forward primer
10.5 l 100 M reverse primer
4 l 180 M probe
225 l H2O

The master mix was thoroughly vortexed and then 416 l aliquots were transferred into eight 2-ml skirted tubes.

Human genomic DNA, 100 g/l (Clontec
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