troubleshoot, help please

I have encountered a few sequencing problems recently. I sequenced the same PCR product (after exoSAP cleanup) in both the Forward direction and Reverse directions that are done in SEPARATE plates. Forward sequence in okay, but there is about 10 to 30 bases that are not sequenced well on the Reverse direction. It first become extremely slanted, and then it becomes "double peak", for example, for location expected one T, it becomes two T, for locations expecting 3 A, it becomes 4 A.

Another weird thing is that the "bad chunks" seems to be around the same region across different samples. I did think about secondary structure, but we have sequenced the same region using the same method previously and this problem just comes up recently. For that, I also ruled out things like annealing temperature, primer design.

Another problem I encountered is premature stop. Recently, most forward reactions stop prematurely after 200 bases (where usually it goes up to 700 to even 900 bases).

Anyone has a clue about the cause of the problems?

Many thanks.

It is really hard to offer help without looking at some example traces. Contact me by email and I will be better able to help.

Daniel Tillett
Nucleics Support