Figure 8

Effect of chain extension time reduction on the Ct values and PCR reproducibility of EG master mix and Power SYBR master mix (ABI). GAPDH was amplified with an EG master mix or Power SYBR master mix using the universal protocol with 15 seconds at 95°C and various chain extension times (60, 40, 20 and 10 seconds, respectively) at 60°C. The two-temperature cycling protocol was recommended by the manufacture (Applied Biosystems) for its Power SYBR master mix. Each amplification was repeated 8 times in order to assess the reproducibility. As the chain extension time was shortened from 60 seconds to 10 seconds, the average Ct value for the reactions using EG master mix increased by about 1 cycle while the standard deviation among octuplicates increased slightly from 0.14 cycle number in 60-second extension time to 0.23 cycle number in 20-second extension time (open bars). Under similar conditions, the average Ct for the amplification using Power SYBR (shaded bars) increased by more than 5 cycles and the standard deviation for Ct increased from 0.10 to 0.43 cycle.