Scientists don’t talk enough about their failures. Trying to learn microbiology from published papers feels like observing the world through a pin hole!
2 years ago I struggled to make dye-based RT-PCR work reliably. I even contacted NEB tech support but they were convinced I had some contamination. I gave up and went to probes, which were expensive and somewhat masked the problem.
Today I learned using a 2-step RT solves all my off-target-amplification problems! It’s more work, but man 45 cycles out and my negative controls show nothing on my gel but primer. Beautiful! https://x.com/RickByersLab/status/1665514755040108551