This can be fairly easily fixed pmlI is a blunt cutter therefore only digest your vector with xbaI/pmlI then digest your fragment only with xbaI. Since fragments produced by PCR already have a blunt end you will be able to do a directional ligation with one blunt end and one sticky end. Please clarify this point " Even the other restriction site that cuts at the same point also present in my DNA fragment?" Do you mean there is a 2nd xbaI site? if that is the case re-amplify your fragment with primers that add AvrII or SpeI or NheI site on the end (choose the enzyme site that is absent internally in your fragment) and then digest only with that enzyme since the end produced by these enzymes are compatible with xbaI.

Hanna Alalam Dear thank you so much for your response, I am also attaching the vector diagram. Actually, I want to digest the highlighted area the GUS gene with XBA1 and Pml1 but the Pml1 site is also present in my gene. even the other enzyme that also cuts at the same point BstEII that has the same issue and its also present in my gene. Thanks. I hope now you can guide better.

ok the solution is simple do not cut your gene with pmlI. Amplify then only cut with xbaI since PCR produces blunt ends. Then ligate the xbaI digested fragment into the pmlI/xbaI digested vector.

Hanna Alalam my gene or vector, means i should not cut the vector with pml1, i just need to digest it with xba1 and then ligate with my gene.

your gene is only cut with xbaI for the vector digest with both

Hanna Alalam But dear why i need to digest the gene, as the gene is already amplified by PCR and for over expression i need the full length fragment.

Sorry i am asking too much. But i am confused so i am asking this.

are you not trying to ligate your gene in instead of GUS?

Yes i am trying to ligate my gene instead of GUS

Hanna Alalam but as i mentioned the restriction sites are also present in my gene, you don,t think they will cut my gene from inappropriate site.

But does your gene contain xbaI?

No my gene dont contain XBA but contains Pml1 and BstEII sites both

Yes then only digest with xbaI (which you added on the primer I assume) and do not digest with pmlI (when you amplfied your gene you get blunt ends) when you cut with xbaI you will get one sticky end on your gene. Now, pmlI is a blunt cutter therefore will give a blunt end on the vector and since the PCR product you made only contains sticky end from xbaI digestion then you do not need to create a blunt end on the other side of the fragment. Then you ligate the xbaI digested fragment into the pmlI/xbaI digested vector this way you are essentially ligating one sticky end and one blunt end (therefore pmlI digestion of the fragment is not neccessary).

Ok now i will just add xba1 site and adapter in my primer desing and remove the Pml1 site.

Thank you so much dear Hanna Alalam for such a great help, i hope this will help me.

Hanna Alalam Hope now you can better understand.