I need to perform an "impossible cloning" as I like to call it, which is inserting a plasmid DNA fragment inside a vector when the restriction sites are not compatible in any way.

Basically, I will digest 2 plasmids with restriction enzymes that produce cohesive ends, since no there are no restriction sites that allow for blunt-ended digestion. One plasmid has the insert I want, the other has the vector I need. Then, I need to blunt both insert and vector, and also dephosphorylate them. At some point, I will also need to gel-purify the correct fragment before proceeding to the ligation.

Here is my expected workflow :

Digestion-->Gel purification-->Blunting (T4 DNA polymerase)-->Column/PCR purification-->Dephosphorylation (Antarctic Alkaline phosphatase)-->Ligation

Is there any way to optimize this workflow ? Please note that one of my restriction enzymes is not heat-inactivable, so a purification step is inevitable I suppose.

I am also aware that I could "simply" add restriction sites in my plasmid by PCR, but I have all the cloning enzymes already so I would rather try conventionnal cloning first.

Thanks !

Martin