I perform a PCR amplification with BSP primers, which have a M13 tail added to a 5' ends. It is a hemi-nested PCR, first round normal BSP primers, second round both primers with M13 tails, and forward primer is a bit closer to the reverse than in the first round.
The amplicon is about 300 bp, and shows a sharp band in a electrophoresis gel. However, when I try direct sequencing for this product (with M13 seqencing primer), I always get unacceptable sequence (e.g. photo attached to the post). It is very confusing, as I succeeded to get good sequences from another 4 amplicons this way. Why is this one different?
I use "Genetic Analyzer 3500" from ABI and BigDye seqencing kit. Any insights are highly anticipated.