I have outsourced Sanger sequencing of my purified PCR products. Looking at the chromatogram, I know to trim the ends. However, the ends that need to be trimmed according to the quality scores are significant portions of the 1907bp gene.
Is this an issue with amplicon preparation or would a different sequencing technology be better for this?
Thanks in advance for any help
Hi Leah Maharaj
typically end of sanger sequencing reactions show lower quality, since fragment are in lower number, approaching the background noise.
Just to be sure on the results, I would cut my by 2 or 3 and produce smaller fragments to sequence.
all the best
fred
Those results are normal. Getting 900+ bases in one read is great. Sequence from an internal primer and/or from the other direction and you'll get high-quality reads to align across the entire product.
In addition to the answers above and on a more technical note, one can't improve the Sanger scores. It is what it is. That means if the quality associated with chromatogram is low, there is no fix for that. In such case, using high quality, pure amplicon without degraded ends should be used and ensure to use correct primers.
Too long amplicons can be tricky with Sanger sequencing.
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