iQ™ Supermix

An accurate one-cycle spacing in C_T is precisely maintained in a series of 2-fold dilutions. Human genomic DNA was amplified with iQ™ supermix, using primers and a probe specific to the IL-1β gene. Eight replicates at each template concentration were amplified along with no-template controls on the MyiQ™ real-time system. r = 0.999, slope = –3.378, efficiency = 97.7%, RFU, relative fluorescence units.

iQ™ supermix and the iScript™ cDNA synthesis kit accomplish accurate, quantitative two-step RT-PCR. Serial dilutions (1 µg–1 pg) of HeLa total RNA were reverse transcribed, and the resulting cDNA was amplified using primers and a probe specific to the α-tubulin gene. Triplicate reactions at each concentration were amplified along with no-template controls on the iCycler iQ^® real-time system. The consistent 3.3-cycle spacing demonstrates the accuracy of amplification with this supermix as well as the fidelity of the iScript kit in reverse transcribing the 10-fold dilutions of the input RNA. r = 0.999, slope = –3.304, efficiency = 100.8%, RFU, relative fluorescence units.

iQ™ supermix provides sensitive real-time detection over 8 orders of magnitude. Tenfold dilutions of a plasmid containing 10⁹ to 10 copies of the α-tubulin gene were amplified using iQ supermix and a FAM-labeled hybridization probe for detection. Eight replicates at each concentration were amplified along with no-template controls on the MyiQ™ real-time system. r = 0.999, slope = –3.365, efficiency = 98.2%, RFU, relative fluorescence units.