wierd dna sequencing results - any clues? (Oct/09/2008 )
hi, just wondering if this has happened to anybody.
ive had a terrible time sequencing some PCR products. The primers i use for sequencing have been used for this before and worked just fine for the guy who started this work (and unfortunatly is no longer in touch).
i have finally discarded that my sample is contaminated via nanodrop and for the the following reason: in some cases i get a really good signal and sequence with my F primer and a terrible one (if any) for the R primer, for other samples (which contain practically the same thing), vice versa, only the R primer works. In some cases both work and in most cases, neither one. When i ask them to repeat the samples, this happens again, but samples that had worked dont work, and samples that didnt work now work.
i have been told this may be due to secondary structures in my sample, which is possible, as i am working with different clinical samples of the same virus, but even so, there must be some way!
any suggestions will be helpful!
Try this website
Most of the time i think sequencing fail or mess up due to the quality of the DNA. Keep em freshly made and dissolved in H20 for best performance.
I get this all the time as well. I spoke to the company that does our sequencing and they said that 99% of the time the DNA is @ too high a concentration. As the other poster said, clean DNA is important, but most folks put way too much DNA in their sequencing reactions. The company said that every time they repeat it, they dilute the sample quite a bit before the run and it usually works pretty well. I would try sending various dilutions of the primer(s) and DNA to find the right combo.
hwo do you prepare your DNA for sequencing and what kind of DNA is it ?
are you doing the sequencing reaction by yourself ?
This company (Macrogen) has some information on why sequencing chromatograms can fail. It might be useful;