That's contamination. Most folks use linearized plasmid as a control instead of PCR products because of the risk of contamination.

Toss ALL of your reagents (and I mean everything, even the probes) and start over from a brand new tube. That includes making new TE buffer, getting a new bottle of molecular grade water, etc.

Use different micropipettes to set up PCR than you use for handling amplified PCR products. Don't set up PCR reactions in the same work space you use to handle amplified PCR products.

Hopefully that will work. If not, you might have to design new primers.

Good luck!

I don't think that the first dilution is ok, a million of copies tend to amplify below ct 20. Are you using a bigger template or are you using the same primers for the template pcr?

As @Katie A S Burnette suggest, try using a plasmid.

Kevin Tran

It seems like you're facing some challenges with the latter half of your standard curve in your TaqMan probe-based qPCR assay. The fact that you're observing no amplification or identical Ct ranges in the later dilutions, along with non-template controls (NTCs) showing signals, suggests potential contamination issues.

Here are some troubleshooting steps you can consider

Additionally, reaching out to the manufacturer of your qPCR reagents for technical support can provide valuable insights.