Dear Abir

i used this kit un the past ( and also the stratagene) for perform single point mutagenesis in genes inserted in pet vectors and it works quite well, however in the last years i Learn the PIPE cloning which u

is an enzime free cloning approach which allow to perform mutagenesis with out and kit and i strongly suggest you to learn this approach.

https://www.ncbi.nlm.nih.gov/m/pubmed/18988020/

you can find more informatio about it on my blog:ProteoCool (https://proteocool.blogspot.com/) at the page1

i'm sure that once you try it, you will never go back to the other old cloning approaches.

good luck

Manuele

Hi Abir.

NEB Q5 is a very good kit, I have used it to make SDM in a plasmid of more than 9kb based on PET and pQE30 vectors. The primers are easy to design on the NEB website. Usually the kit comes with a high fidelity DNA polymerase or you can get it separately. I have had very good experience with Phusion DNA polymerase for SDM. The protocol is very simple, practically in two days you can obtain the mutated plasmids.

I'm sure it will work for you.

Victor

following

I strongly recommend it. We have used it with a lot of success. As with all systems, particularly the ones you use PCR amplification, sequence the whole gene to ensure there are no unwanted mutations. Not often, but we have seen it. If you have a problem, it is most likely the primers (and you did not get enough amplification, which you can check with a gel). The good thing about this procedure is that there are no many "moving parts" or steps. Very easy to do truncations or deletions.

You really don't need a kit to do SDM. A high-fidelity polymerase (Phusion or Q5) and DpnI are all that's needed (and T4 ligase if you do back-to-back primers). The primers you have to make yourself anyway, and you can easily make competent cells etc. yourself too. You can skip the T4 ligation step as well if you make partially overlapping primers (e.g. doi: 10.1186/1472-6750-8-91 - this protocol has worked very nicely for me).

Thank you all.