Try cutting your plasmid to linearize and then repeat the PCR. Sometimes the coiled structure of pure plasmid will interfere with PCR. Make sure you choose an endonuclease that will not make a cut in your inserted DNA region.

Good luck!

If you said that you did not get the correct sequence from your plasmid preparation, then you should not continue to use that plasmid prep. I would go back a step and retransform, screen, and purify plasmid DNA from a retransformant that has the right PCR profile, then sequence again. You might have had a mixed population so you got the proper PCR product from a sub-fraction of the population but not the overall correct sequence from the plasmid prep.

Most sequencing companies have recommended Tm for sequencing primers to be sent along with the PCR product. If the gene specific primers are not working well in the sequencing reactions, the problem might be to do with the Tm of these primers

Your colony PCR might be positive because of mixed population. It is very difficult to isolate the pure colony harbouring the correct plamid with insert. Instead of wasting time for identifying correct colony, I suggest you to retransform and repeat the entire procedure. Once your purified plasmid DNA is positive for PCR using gene specific primers, send it for sequencing.

Pendru Raghunath Hello thanks for your suggestion. As you have suggested "Once your purified plasmid DNA is positive for PCR using gene specific primers, send it for sequencing." I did the PCR using the purified plasmid and got the positive PCR band. I sent that plasmid for sequencing and PCR product also. But in my case PCR product gave the correct sequencing result but not the purified plasmid using gene specific primer.

Michael J. Benedik Thanks a lot for your suggestion . I will try to do the same.

Sarah Nanyiti Thanks I will keep that thing in my mind.