I am trying to sequence a PCR product, the length is sufficient, it on agarose gel it gives nice, sharp band, without any smear or clouds of leftover oligos. Concentration measured on spectrophotometer also seems to be okay. What is interesing - fluorymetry specific for dsDNA shows only about a fraction of the spectro values, but anyway the concentration shown should be more than ok for sequencing. I do not get heterogeneous read, sequence is basically unreadable and the signal decays quickly, looks like classic problem but it isn't. We used same chemistry on other samples and they were fine. Secondary structure - rather not, as it would cause problem with PCR before sequencing. Any hints greatly appreciated.
Have you tried sequencing from an internal primer or from your forward and reverse primers? Sometimes a primer that works well for amplification does not work for sequencing.
This doesn't look like primer fail, signal decay is quite obvious, also these primers were already used for successful sequencing. We tried with both ends (F and R).
can you post the .abi file of the sequence please these files are often informative. You can not be entirely sure about secondary structure as sequencing mostly happens at lower temperatures than the original pcr. Also what separation method are you using to get rid of your primers from the template prior to sequencing
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