Dear Dr. Donia,

DNA template concentration might be too low so you can check the concentration of your template to ensure that it falls with the ranges listed in the Sample Submission Guidelines

Low quality preparation yields incomplete removal of protein and/or RNA, or buffer salts or other chemical contamination may be present. Any of these conditions can inhibit sequencing reaction enzymes.

So,you can improve the quality of the template prep by using a commercial mini-prep kit and by preparing fresh stock solutions for template prep and dilution just prior to submitting

samples for sequencing.

I hope this might help.

Thanks, Dr. Rasha for your answer

I already used miniprep kit for isolation of plasmid followed by phenol-chloroform and ethanol precipitation to increase the purity of plasmid then PCR using different primers and ethanol precipitation for the preparation of the sample for sequencing. Also,   I have decreased the concentration of plasmid DNA  to be  100 ng instead of 200 ng. 

Best Regards 

some reasons for no signal will be no template, incorrect primers and annealing temperature too high for the priers to anneal. I would try sequencing with the pcr primers to ensure the quality of kit ,ocr machine parameters and post sequencing purification method and if this works then I would assume that your sequencing primers are degraded and get them remade. If it still does not work consider thet for this plasmid there is no sequence homology with your sequencing primer. Include a positive sequencing control if one comes with the kit and if you have an in house sequencer check if any/all other users have the same problem in case the sequencer is faulty

Thanks so much  Dr. Paul  I really appreciate your response you offer all possibilities 

Best Regards 

Thanks a lot,  Dr. Mohamed actually we thought  about this idea today in lab and we are going to perform it soon

Best Regards