I use sanger method regularly to sequence DNA but this time I came across a problem which I couldn’t find any sense
I did sequence different segments of DNA double strands that belongs to different genes and all showed great results in the Reverse strand, but there was no reading or any useable results after sequencing the F strand, anyone have any Idea?
Check whether the complementary sequence for the forward primer is in the DNA as sequenced by the reverse primer. If no, design a new primer. If yes, perhaps the primer might be A/T-rich and the annealing temperature used during sequencing is too high.
Thank you for your answer and perhaps it would be good to say that the same primers have been used many times before, we had no such problem regarding the design or the location or the temperature.
Hmm, since the primer has worked in the past that rules out a few possible answers. My best guess is that the primer has degraded (it happens).
My advice would be to make a fresh dilution of your primer from the frozen stock & try again.
Good luck!
- Badges
- Science topic