Hi Daniel, I used the primers I used in PCR to generate the PCR product. On the dyes, am not sure since i sent my samples to a sequencing company, unless I find out. However, the readable part corresponds to my expected sequence.

Hi Elisabeth, I would say you are using too much of the reverse primer, for this reason you start too many fragments that terminate early and you use up material before you can get longer readings. You were luckier with the forward primer, but the behaviour is similar: you see the decrease in the peaks starting already at around 350 bp, it's a bit early (if you had to read 900-1000 bp as normally this sequencing method allows, I don't think you would have been able).

Normally the sequencing of PCR fragments works very well with 100-150 ng of DNA for every 100 nt to sequence (so, in your case 500-750 ng of PCR product) and 3-4 pmol of primer (1.5-1.7 microliters of primer diluted 2 micromolar)...

Thanks Daniel and Pietro, I will put your input into consideration and see how it goes. Thanks alot.

Hi Elizabeth: I suggest first check quantity of DNA you get after purification before going for sequencing PCR. By looking at electroencephalograms you uploaded, I think  try to excise DNA bands from gel, purify and sequence; we resolved our similar problem using this approach. Using alternative reverse primer internal to you already used might work also. Be sure to use any new primer for PCR amplification, before using for Sequencing PCR.

Hi Elizabeth, did you quantify your PCR products before sending for sequencing, you will need between 2.5ng-5ng PCR product per 100bp. In addition, did you record multiple bands on your gels, if there are multiple bands, you might need to purify your product using gel elution.