Hi Julie - in order to answer your question better, I could do with a bit of clarification. As I read your question, it sounds to me like you want to remove an inserted DNA fragment from a plasmid by digesting it with the restriction enzyme EcoRI, then you'd like to stick the plasmid back together (without that DNA insert fragment) and amplify it.

If that's the case then it's a fairly straightforward process. I would take 1 µg of the plasmid, pop it in your restriction digest reaction with EcoRI and the relevant buffer (total volume to 20 µl with sterile water) for 1-2 hours at 37°C. You can then run the entire reaction on an agarose gel alongside 500 ng or 1 µg of the original undigested plasmid to check that it's been linearised and your DNA fragment has been successfully removed. Then cut the plasmid out of the agarose gel (making sure to choose the DNA band of the right size), purify it and set a ligation reaction up using 100 ng of the purified plasmid. Transform the ligation reaction into E.coli DH5a cells and then you can perform colony PCR, or do minipreps and then PCR and sequencing to confirm that you have your desired plasmid which now lacks the unwanted DNA fragment. If successful, you'll then have a stock of your plasmid (a miniprep). There is no need to heat inactivate the EcoRI enzyme during this process, because you're going to gel purify the DNA anyway and that should remove/denature the enzyme.

I hope this helps (and apologies if I have misread your question entirely)!

It can be done as Jonathan Whitchurch mentioned, but I don't see a reason to do that. Cloning is done to amplify insert. If you want to remove the insert, why would you want to amplify an empty vector?

Dear Julie

i also do not undertand way, if you would amplamplify a fragment from a vector, you need to digest it in Advance.you can simple use the sentire vector as dna templat of your pcr.

ciao

Manuele

you can simple amplify with pcr ( ho fidelity polimerase) the all the vector but not the fragment that you would like to remove with an approach similar to the one used for single point mutagenesis and after close the vector by digestion location Thanks to an enzima that giù introduce into the primers or otherwise by PIPE cloning by addiction of an overlapping ragion to your primer.

you can find more details regarding PIPE cloning on my blog: https://proteocool.blogspot.com

good luck

Manuele

Thank you all for the reply! Maybe I wasn't really clear but I would like to remove a small section of my insert. So not my entire insert. I will try to do gel purification.

Thanks again

kind regards

Julie

Dear Julie.

if the fragment to be removed is just one and not 2, there is no differences beetween remove the entire insert or only a c-terminal , n

-terminal or internal portion, it just depends to how you desing the primers to perfom the v-pcr

give a look to

https://onlinelibrary.wiley.com/doi/full/10.1002/prot.21786

ciao

Manuele

Julie Jacobs Why do you want to take so much of trouble of restricting and reusing that plasmid? Why can't you d cloning with the insert which you actually want to make sure you get what you actually want. I suppose this would be easier and cheaper way towards a good result.