Hello,
I have problems with plasmid and PCR fragments sequencing (Sanger sequencing on SeqStudio device). We perform sequencing reaction with BrilliantDye™ Terminator v3.1 with subsequent purification on iX-Pure™ DyeTerminator (both from Nimagen).
Sometimes we have poor reads; for example, sudden signal drop or loss of peak resolution in the middle of the run. When we repeat the run within few hours (same sample, same position on the plate, i.e. same capillary) we sometimes have much better reads. On the other hand, in some cases second run produces poor quality chromatograms as compared to the first one (but this may be attributed to sample degradation). Please see examples attached.
It looks like there is some problem with sequencing device, not with the reaction mixes. Has anyone experienced this kind of situation? What are the reasons and how to solve this problem?
Thank you in advance for your help.
Stan