A simple solution would be to design primers flanking the GFP insert so that a PCR amplifies the vector without the fluorophore. You can add unique restriction sites to the primers, so that you may easily insert your new fluorophore into the generated construct. Use the same approach to add these restriction sites to the mCherry cDNA to allow ligation.

Cheers,

Jan

You may design the suitable primers with the GFP insert so that an amplified vector without the fluorophore

thank you Jan Philipp Dobert for this logical solution ! but the thing is that I have a relatively big vector with more than 6000 bp, and my question now , is it actually possible to amplify a whole plasmid by a regular pcr reaction? I mean then I just have to increase the elongation time , right?

This should not be an issue, we do this regularly in our lab. Just make sure to use a high-fidelity polymerase (Q5 is what we use) and set an appropriate elongation time.

Good Luck!

Ilango Sakkanan sorry I didn't understand, why should I include GFP in my primers ?

@Jan Philipp Dobert thanks ! I will try that.

You could use Cas9 for digestion if there are PAM sites in the correct places.

A partial digestion with the non-unique enzyme may work, but it will be very inefficient.

PCR seems to be the best approach. If the amplification of the whole plasmid does not work out, you could amplify fragments of the plasmid using overlapping primers and re-assemble the plasmid using Gibson assembly.

Aidas Nasevicius thank you for your answer ! that is really helpful.

Are there any other sites flanking the GFP? A plasmid map would be helpful to think of other cloning ideas.

How did the GFP end up with BamHI sites if BamHI is not unique in the vector? Was something else added to the vector after the GFP to add the other BamHI location? Or was the GFP-gene added via other sites?

If you are looking for creative solutions, I would caution against partial digests. Restriction enzymes cut really quickly and you'd end up with a lot of unwanted digest products.

Amy Klocko thank you for your answer. the problem is that I had this vector from another group and we both don't know the plasmid map, we could just know what the insert is. I digested the plasmid with BamH1, and because of the many digestion products I knew that BamH1 restriction site was not unique.

Hmm, no map is going to make your job really tricky. At this point it may be easier to build your other fusion in a nice known plasmid.

Or, if you know (and can verify) which sites flank your gene you could remove the entire gene GFP cassette and swap in one your built in a subcloning vector.

Just to clarify - is this an internal GFP tag or a terminal fusion tag?

Amy made an important point here: Not knowing what vector your construct is based upon is not only tricky on an experimental level, but also when you ultimately want to publish your data.

You could find out what vector you have via sequencing with custom primers and BLASTing your results. But at that point, you might as well assemble a new construct with a known vector, like Amy suggested.

Cheers,

Jan

What Amy Klocko said - transferring into a new vector or at least sequencing your plasmid would help you a lot. You probably will save a lot of time, effort and money downstream. And will avoid a good amount of head scratching while looking at your gels and wondering what is going on.

actually I know the gene sequence (the insert), and the GFP is an internal tag.

and right now I'm transferring the whole insert to another known plasmid, but my problem is that BamH1 restriction site. it's not unique on both of the vector, and it is really important for me to replace this GFP with another fluorophore.

thank you for the suggestions =)

Hadeel Khalouf Hi dear Dr Hadeel. was you able to do that replacing?