What are you using to dilute your purified plasmid? I found out that the elution buffer was causing issues when I was trying to sequence because the EDTA in the buffer was interrupting the machine read out. After I started diluting with purified MilliQ water I stopped having issues.

Actually, the plasmid was ontained from a maxi-prep and was eluted in TE buffer. Before sequencing I diluted to 100 ng/uL in esterile miliQ water, but there could be EDTA yet in solution

I will try to eliminate it completely somehow, and give it a try

Thank you so much for your answer!

Unless this section of the chromatogram is from the very end then I don't think you had a good sequencing reaction at all. Those do not look like particularly good peaks and if you notice you have really high levels of crossover signals in many of the peaks.

That is the problem, this is the very beginning of the chromatogram. I am trying to find out what has happened. I am trying now with a different primer that has given me good results in the past, to see if the primer was the problem.

Thankyou for your answer

Post the entire chromatogram pic, that might help others diagnose.

Thank you, You are right, may be the entire chromatogram can help. I add here a zoomed out vision of the entire result.

News:

- I have tried other primer that usually works and I have had no result

- I have changed now the capillary and used a new bottle of the POP6 polymer and waiting for the result.

Any way, thank you so much for your comments

Kind regards

Someone else may be able to diagnose it better than I can, but to my eyes this is a failed reaction pretty much the entire length. So its not like it started well and then failed.

Dear Michael,

I really appreciate your opinion, is very valuable for me.

I have tried everything related with the equipment, I have changed tha capillary, used new polymer, different primers and plasmids and i always have a very short reading. It seems that you are right, the only thing I have not tested yet is new Big dye mix.

What I have noticed in all these runs is that in the first part of the chromatogram there seems to be some good looking peaks (at least it seems) but in the last part, the peaks turns more flattened or poor resolved or something like that.

My next step y buy a new big dye kit and try again.

Again, thank you so much

Regards

I'm not sure the problem is the big dye kit, more likely the problem is the reaction or the cleanup. Why don't you try with a known control DNA and primer that you KNOW will work for certain.