I received my sequence data for the plasmid with my gene of interest, and when i am trying to get annotation, my forward primer plasmid does have four mismatches with the gene of interest, and the reverse plasmid does have one mismatch with GOI. What is the reason and can i go for downstream applications?
Actually, I need to know several points such as why you sequenced this plasmid and what kind of seq data it is.
1- If you do sequencing to see whether your insertion successful or not, getting several mismatches can be fine as you can see gene is already inside the plasmid. Other point is that I think dont worry about the mismatches in primer sites because if there was a real problem in those sites, you would not get a seq data at all.
2- Secondly, if it is Sanger seq data it is normal to have such errors as the technique gives error often by itself, in primer sites particularly.
Finally, I think you can go for downstream applications, if you do simple cloning.
@hasan i need to make sure sequence of my gene of intrest in the plasmid , mismatch are not in the primer sequence , i found four Mismatch in the forward sequence of my gene of intrest and 1 mismatch in the reverse sequence of my gene of intrest.
@Kyle thank you very much for your answer , yeah my sequence is from more up to date genome and i also checked about silent mutation but ots not silent mutation .
@Kyle thank you very much for your answer , yeah my sequence is from more up to date genome and i also checked about silent mutation but ots not silent mutation .