Protocol Online logo
Top : Forum Archives: : Real-Time PCR

Decreasing Amplification Curve for Standards - (Feb/12/2006 )

Hi all,

I'm trying to quantify bacterial 16 S genes from environmental samples using SYBR Green on a iCycler. In my amplification curve, the amplification curve for the highest concentration standards increases, crosses the threshold, and then decreases in subsequent cycles. Any ideas why this is so?

Thanks a bunch for any input you have!

(I've attached the curve for your consideration)

Attached Image



I would guess an oversaturation of the system, but I can't see how this would work with sybr-green, unless there is so much binding at the later stages, that there is competitive inhibition of the fluoresence.



Thanks for your reply, Bob. Here's some more info about it: I ran the qPCR products from the standards out on a gel, and there are smears for the highest concentrations.