Hello,

So in my lab we've been doing site-directed mutagenesis for a long time using the same kit (Agilent's Quick Flash) and it's always worked wonders, but a few months ago I did one and when looking at the results, instead of having my insert with the mutation I wanted, I had none of my original insert and instead a part of the gene SMG5 was inserted in its place.

At the time I thought it was weird but I had more important stuff to attend to, and since I thought it might have been some contamination in the kit and it just ran out when I used it, I put the whole thing aside.

That's until a few weeks ago when a lab mate ordered a new set of the same mutagenesis kit, and surely enough, got the same result of the same SMG5 fragment being inserted and her original insert disappearing, even though she was working on an entirely different insert, but with the same vector as I did. That made me think, and I looked at the last mutagenesis that had been made by someone else like a year ago which had also failed, and again, the same insert was showing up.

We are gonna test it but still that pretty much rules out the kit being the source of the problem since it was a brand new one and still gave the same issue as the one I used, and the person who did it a year ago used an entirely different kit that wasn't even the same type as the other two cases. We also change stuff like liquid LB and other material regularly so it's pretty difficult the contamination comes from there.

The other thing in common is the original vector we cloned into, but since after cloning you specifically select a clone that's supposed to have just one specific plasmid, and we tested those, and saw none that would have integrated that SMG5 insert prior to mutagenesis. It's only when we do mutagenesis that this happens, no other issues cloning anything or doing anything with the plasmids otherwise.

Any ideas what could be wrong or anyone who has ran into the same or a similar issue?