Can you add an original .abi sequence file please?

Ns are normal both at the primer end where sequencing is poor for the first 49 bases and also at the other end of the sequence where the signal strength becomes weaker and background signal becomes comparable with the sequence electropherogram. After the expected end of the sequence a sequencer will still try to fit the background signal into the sequence and there will be many Ns. If the forward sequence is almost all good and the reverse is almost all bad then you may have an insertion/deletion near the reverse end of the sequence (or a poly base or CA repeat) and from that point all bases will have 2 bases at each position and will look like a real mess. It would be helpful to see the original files.

Are you using exo-sap as the method of cleanup of the pcr product?

I agree with Paul Rutland that this could be an issue with heterogeneity of the sequence. But if you see the same thing with 10 clones it seems to me more likely that there is some systemic problem, maybe your stock of reverse primer has gone bad or gotten contaminated with some other primer, or the reverse primer is not really working well on the plasmid you are using. I would suggest some control experiments, what happens with a control plasmid with no insert?

Michael J. Benedik and Paul Rutland let me add two sequences the forward and the reverse of one isolate. Probably they may shed some light

This seems pretty clear. With the forward sequence you have clean sequence up to about 510 nts and then the sequence goes really bad as if there was some event like a deletion around 510 in some of the molecules such that you have a mixed or heterogeneous population after 510. So with the reverse sequence you would only be reading the mixed section. This is what Paul described.

I would suggest taking your plasmid preps and retransforming at very low dilution and then picking some single colonies to prep and resequenced.

You might also look at the sequence of your clone at the 510 position and see if there is something obvious at that location that might explain it.

Just a pointer i am working with pure bacterial isolates.... I don't know if this makes it any different....... Besides i have done other genes on the same and the sequences always came out ok

are you cleaning the pcr product prior to sequencing with exo-sap Edwin Wanjofu ?

Paul Rutland yea i do cleanup first using Exo-1 and FASTAP

Good then here is a possibility. Your forwards pcr /sequencing primer has a motif like GGGG or ggnncc and is capable of G quadruplex or even just forming a primer dimer with other forwards primer molecules or with itself. These complexes now have a double stranded component but exosap can only digest single stranded dna so after the cleanup stage you have removed the reverse primer but retain a shortened forwards primer. When you add forwards sequencing primer both it and the shortmer anneal at the same 3' position so give you a good sequence with quite strong peaks but without the very large early dye peak indicating correct sequencing mix reagents.

When you sequence in reverse ,however, you have not removed the shortened forwards primer due to exosaps inability to degrade it but now you add a sequencing reverse primer. Your sequencing reaction now has primers at both ends so you get 1 2 sequences which looks messy as expected and

2 a huge early dye peak because you have far too much primer which uses up all of the dye terminators early in the reaction so giving a strong early dye peak an leaving little dye mix to be incorporated into the sequence peaks which are much weaker.

This possibility is primer sequence specific so I have no problem with all of your other primer sets working well as expected

wow so informative..Thanks so much Paul Rutland

you are welcome Edwin Wanjofu . A simple test of this idea is to exo sap a sample. DO NOT add sequencing primer and run the sequencing reaction and astonishingly for the rare times when exosap has a problem you will get a nice clean electropherogram. all the best,Paul