I just purchased the single channel qPCR machine and tried running some samples after calibration. However, I was unable to detect any amplification curves using the software. I used DNA and the Chai green master mix. The point of the qPCR is determine how many cycles I should run to amplify Illumina DNA libraries for sequencing. I thought I should have at least seen primer-dimer amplification but the plots were blank. Any ideas of what I may have done wrong?
(1) 1µl of DNA Pre-Library
(2) 0.75µl of 10uM of indexing primer i5 (F primer)
(3) 0.75µl of 10uM of indexing primer i7 (R primer)
(4) 17.5 ul Chai Green Master Mix (2x)
Program: initial denature at 95°C for 5 min, the following (98°C for 20 s, 60°C for 15 s, 72°C for 30 s) for 30 cycles, final extension at 72°C for 5 min, hold at 4°C.