The problem we faced with is the failed emPCR - less 2% output. Increasing of matrix input didn’t improve situation.
Our libraries (for A-lib kit) were prepared by 2-step PCR. A-key, B-key and MIDs were attached to the target amplicons during second step.
We sequenced our libraries by Sanger and found that there are additional unexpected nucleotides in the ends: AG on the B-key and A on the A-key.
We wonder whether these unwanted nucleotides appeared due defective primers we got or inappropriate polymerase we used (Tersus) or may be they are just sequencing artefacts (Applied Biosystems BigDye® Terminator v3.1).
And the main question is - How such nucleotides could affect emPCR?
(another version is a bad emPCR kit)
It would be nice to hear your considerations.
Or maybe someone had similar difficulties.