Protocol Online logo
Top : Forum Archives: : Molecular Biology

Help. Trying to sequence my plasmids, but the primers refuse to prime! - (Jan/08/2008 )

I have been struggling with these clones for months now. I have received new aliquots form the supplier. When I send them off for sequencing, none of my primers will anneal, and I get no sequencing results. I have tried GS primers and 2 sifferent aliquots of the VECTOR primers provided by the company who supplied the clones. The supplier told me yesterday that they have had some problems with the vector primers not priming (they only work 40% of the time). My questions, is "what makes the primers not want to prime, or anneal?" My A260/280 values are excellent, my DNA concentrations are good, there is no salt contamination, and the primers have worked previously with past samples. What could be happening? Thank You.


we have had similar problems with primers not giving us signal in sequencing. The sequencing center as usual gives us a whole lot of reason, but honestly, I am not sure !!!

We designed primers just a few bases up or downstream of the original primer and they work.