Are you saying there aren't useful endonuclease sites around the sequence you want to remove? One alternative would be try inverse PCR'ing the rest of your desired plasmid sequence then phosphorylating & ligating the product. It's a big plasmid so the DNA polymerase might struggle (so another alternative would be to amplify your plasmid in chunks then assemble them by Gibson cloning).

Are you saying there aren't useful endonuclease sites around the sequence you want to remove? One alternative would be try inverse PCR'ing the rest of your desired plasmid sequence then phosphorylating & ligating the product. It's a big plasmid so the DNA polymerase might struggle (so another alternative would be to amplify your plasmid in chunks then assemble them by Gibson cloning).

Hi Tasnim, I would agree with Cason about using PCR. I have successfully used the NEB Gibson assembly protocols for something similar, and would recommend it.

I agree with the two previous answers. The mutagenesis kit of New England Biolabs (I own no stock) provides a mix of kinase, ligase, and DpnI njuclease (to destroy the template) that works quite nicely to religate the PCR product. It is advisable to check the amplified product by agarose gel electrophoresis and possibly gel purify it in order to avoid unwanted by-products.

If I undesrtand correctly, you would like to excise 2kb from a ~11kb plasmid because it encodes a protein you dont want?

But surely there is a unique restriction site within that sequence that will generate sticky ends that you can fill-in/SS nuclease remove, then religate? That should throw out the reading frame and render the protein inactive.

Hi Tasnim,

If you have the plasmid DNA sequence, just run a few bioinformatic tools and detect all possible restriction sites. I hope definitely, there should be a suitable restriction sites flanking your protein gene. If there is no site, then probably you will have to try other methods prescribed above by other researchers.

Thank you.

Hi Tasnim,

the question is not so clear to me as well, but as far as i understood if you are looking to remove that 2kb DNA stretch from the plasmid, you can simply use the sequence and digest it in silico and then select the appropriate enzymes to digest with and then ligate the desired fragment. and if you don't find any such restriction site you can opt for infusion cloning or gibson assembly, since the size of the plasmid after removal is around 9 kb you can assemble it 4 fragments. that might help you.

if it is only about the protein i.e if you want to knock out that gene, then i recommend CRISPR-CAS9 mediated gene knock out.

Hi Tasnim,

If you can't use classic approach involving restriction enzymes and DNA ligase to re-engineer your plasmid. I would use four primers PCR (SLIM) described by Chiu and colleagues;

https://academic.oup.com/nar/article/32/21/e174/1101874

I have been using it successfully for years to generate insertions and deletions in plasmids. From practical point of view, SLIM protocol is as efficient and straight forward as site-directed mutagenesis procedure.

All the best,

Stan

Thank you for the answers. I think i understood what needs to be done. I will be using PCR to remove the unwanted gene sequence and amplify the rest. Thank you all for you suggestions. It was really helpful