Hello,
I am designing a Taqman assay and have used ABI's Primer Express software to design the primers and probe. I've also had a bioinformatician check that the sequences are in conserved areas in the genome.
I've done a gradient to determine the best annealing/extension (combined) temperature to use. After finding the best option, with the best amplification efficiency, and no amplification curve on my negative controls, I ran the product on a gel. I am seeing nonspecific bands in some of my negative samples, at higher mw than my target. My positive control is at 146bp.
I've tried using the second best annealing temp, but still seeing the same. I've also lowered the annealing/extension time from 45sec to 30secs, and the band/s are still there.
What else can I do? Would having separate annealing and extension temps help?
Thank you in advance.
- Badges
- Science method