Can you attach an original .abi sequence file. The nature of the noise in your sequence may give clues to the origin of the poor sequencing

Paul Rutland this is the plasmid extracted from one of the colonies.

Thank you Michele Ferro I do see the result of your problem. The unincorporated dye peak is low and the signal intensities start at an acceptable 1200 AFU but drop to a poor 300AFU much too soon. So it seems that the chemistry is good quality ( as you would expect from MWG) but the peak separations are overlapping which can be caused by having multiple templates or multiple binding sites for your sequencing primer or mixed sequencing primers or poor quality sequencing primer ( too many n-1,n-2 sequences in your primer. or an indel close to the sequencing primer creating 2 sequences of different lengths..

If this result was from plasmid with insert I would check that the plasmid was a single clone, that it only contains one sequence and that the sequencing primer can only anneal at one position in the plasmid. The fact that you do get sequence over 700 bases suggests that the amount of primer and the amount of template that you sent MWG are correct . If this result is caused by an indel or poor quality sequencing primer then sequencing with a reverse primer may look better.

good luck

Paul

thank you Paul Rutland , i actually went through two steps of purification. I transformed some competent bacteria with the DNA from the prep and selected single colonies to be screened, but it is still noisy. I will definitely look into the primers and try different ones.

Thank you so much for your imputs!

Michele

It might be worth a restriction digest of the template plasmid. A single insertion or single species of plasmid should produce a simple band pattern and anything more complex might indicate multiple inserts or templates