gel electrophoresis - (Jun/26/2014 )

Dear all,

I am trying to remove a part from my plasmid.

The size of this part is +- 200Bp.

Is it needed to put my sample on a gel after I digested the vector ?

The only reason I see is to remove that small part from my vector so it does not get ligated back into the vector, but would this happen if I transform e. coli right away with the digested vector?

A different reason is that you put plasmids on a gel is in order to remove bigger parts, to make sure you work with the right part, but here its almost impossible to distinguish between digested vector and non digested vector (vector is 9800 bps, so removing 200 wont make such a difference, I doubt I would see it on a gel)

I wouldn't put it on gel, but I would try to counteract the transformation of the original vector (either because it was not digested or because it was only cut once and religated during the ligation) by incubating the ligation mixture with a restriction enzyme that only cuts in the 200 bp fragment you want to remove. This way, you will linearize all 'original' vectors and should only end up with the ones lacking the 200 bp fragment.

Yes, thats an idea, I'll check if I can do this.

thanks.

A different question: after doing this extra digest , would you than ethanol precipitate your DNA  ? or not? I think it does not matter for the transformation, but I am not sure.

dpo on Thu Jun 26 11:09:07 2014 said:

I wouldn't put it on gel, but I would try to counteract the transformation of the original vector (either because it was not digested or because it was only cut once and religated during the ligation) by incubating the ligation mixture with a restriction enzyme that only cuts in the 200 bp fragment you want to remove. This way, you will linearize all 'original' vectors and should only end up with the ones lacking the 200 bp fragment.

When I did this, it depended on the kind of transformation: when I used it for electroporation, where I added a very small volume of the ligation mixture to the cells, I precipitated it. But when doing transformation by heat shock, where you can use a larger volume, I didn't bother. I don't remember if I heat-inactivated the enzyme before the heat shock (it's been a couple of years since I did my last transformation), but it's something you might consider.

I would add the restriction enzyme directly to the ligation mix, incubate for 15 minutes, and directly transform.