Hello,
I have problems with PCR fragment sequencing (Sanger sequencing on SeqStudio). We perform sequencing reaction with BigDye™ Terminator v3.1 with subsequent purification with BigDye™ XTerminator. Sometimes sequencing reads generate spurious peaks. Please see examples attached. Has anyone experienced this kind of situation? What are the reasons and how to solve this problem?
Thank you in advance for your help.
I would separate your first picture from the second and the third, as they represent different issues. On the 1st pic, timing is slightly off, need to adjust/determine the peaks manually. On 2nd/3rd pics, one dTP is too much - may be expired reagents, multiple amplicons, not uniform amplicon size, or the machine needs calibration
I agree with Péter Gyarmati , the second & third pictures are runs where you probably don't have enough template to generate any data. That sort of "static"/many overlapping wiggly lines means not enough template. The overlapping peaks in the bottom half of the third picture can also be from having a mixture of more than one template molecule.
You can solve this by running out an aliquot of the template molecules on an agarose gel to check the band size & intensity. Faint bands don't give good sequence data. Also, make sure there is just 1 band.
Thank you, Péter Gyarmati and Katie A S Burnette, for your responses. I appreciate your insights.
I wanted to inform you that I have conducted gel agarose electrophoresis after amplifying the MLST genes, and the bands were good.
Do you recommend performing another gel electrophoresis before proceeding with Sanger sequencing?
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