Posted 23 February 2009 - 12:34 PM
Hi there, I have been strugling through several primers and probes to detect leptospira in urine. I decided to go for Taqman probe and primers. The curves were pretty good but the efficiency was pretty low. So I kept the probe and I designed primers with Primer3 instead of primer express. The efficiency came out pretty good but now the curves dont look sigmoidal at all. Also with this longer primers not designed by the Applied Biosystems software, the detection level is better, the sensitivity has increased dramatically, but as mentioned before, the curves never reach Plateau. I was wondering if anybody has experienced the same problem. The primer concetration I am using at the moment is 600nM and the probe concentration is 250nM. I got this concentrations after optimization. Thank you very much.