I've been encountering several errors when working on my relative-qPCR. My project uses two different assays to assess telomere length against a single-copy gene using SYBR as the dye of choice.

Master Mix

SYBR Green Master Mix (2X) - 12.5 uL

Forward Primer (3uM) - 1 uL

Reverse Primer (3uM) - 1 uL

14.5 uL MM + 10.5 uL template DNA (5 ng/uL)

Run Conditions

Telomere

95 10:00 Hot-Start

95 00:10 Denature

62 00:20 Anneal

72 00:15 Extend

+Plate Read

x40

+Melt Curve

Single-Copy Gene

95 10:00 Hot-Start

95 00:10 Denature

55 00:20 Anneal

72 00:15 Extend

+Plate Read

x40

+Melt Curve

Efficiency

I am using LinRegPCR to set the baseline threshold from raw fluorescence data and to determine efficiency within each well. Currently, primer efficiency is around 1.4-2.0. I suspect this is because of the large amount of fluorescence occurring during the earlier cycles.

NTC

Even after replacing the autoclaved MilliQ Water, the NTC appears to have become contaminated as evidenced by a high Cq (~26) and a melt curve similar to the samples. I suspect that the working primer stock may have become contaminated.