this link is useful

https://www.ncbi.nlm.nih.gov/books/NBK21498/

regards

Hello

Digest the plasmid using Cutsmart buffer overnight at 37°C, then purify the open plasmid after electrophoresis on agarose gel using nucleospin kit or equivalent

Dear Nkemngo,

you have correctly identified this as a potential problem. The plasmid map on Addgene shows that the restriction sites are right next to each other, but not overlapping.

Also, if you check https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

you will see that XhoI and BamHI will cleave reasonably well with only 1-3bp in addition to the recognition site.

So my recommendation would be to do a sequential digest - one enzyme, then inactivate or purify, then do the second enzyme. Hopefully that should give you properly digested plasmid.

If this doesn't work out, I would digest with NdeI and BamHI and design a primer pair including an NdeI site to amplify your intended insert and do the cloning that way.

Let us know how you get on!

Hello

I am most grateful for all the contributions.

Regards,

Dear Petra Disterer,

Thank you very much for such an explicit answer. I would definitely keep you updated on the progress of the cloning exercise.

Regards,