I am facing a problem with a new Primer set I designed and I hope that someone can help me with this.
I designed primers for SYBR Green qPCR and ran a PCR with temperature gradient from 57-62°C.
The Primers are blasted and are specific for my sequence. One splice variant is also amplified which has the same amplicon size.
The no template control and no rt control did not show any amplification.
I see a broad peak in the melt curve of my product at many temperatures. At some temperatures the meltcurve looks good to me. I ran the products on a 2% Agarose Gel.
Multiple Bands larger than my specific product (120bp) can be seen on the Gel.
Is there anybody who can help me out with this?
Best regards Alex