Plasmid-self ligating - (Sep/14/2005 )

Hey all!
My task is to cut a circular plasmid with two enzymes and ligate cDNA to it. When I cut with the first plasmid, there is definately linearisation, so I know that enzyme has cut. After I cut with the second, I gel elute to prevent any self-ligation.
I always set up a control for ligation to see if there are any colonies due to self ligation. and there are loads!
To make sure the second enzyme has been cutting, I have set up a digestion with that first and the plasmid linearises, so I know that enzyme is not inactive.
The same thing happened with another plasmid as well, so we decided to switch over to another one, thinking it would be easier. But no use.
Please help!

hi
basically, you can dephosphorylate your vector and increase the ratio insert/vector.

Thanks Fred.
But I should have mentioned the enzymes that I cut the plasmid with yields sticky ends which I require, so does dephosphorylating really help in this case as well? I know that its very helpful when the plasmid has blunt ends.

hi
even if you got non compatible sticky ends, plasmid can self religate. Obviously less than blunt end but it's possible. I've got once in a religation step a change of 1base that killed the restriction site... so everything can happen with living...
fred

How close together are the restiction sites? If you cut with one enzyme, then you'll have a linearized fragment...however, if the second site is right on the end of the linearized fragment you will have a problem. Restriction enzymes like to have a certain amont of DNA on either side of the restriction site to be 100 % active. What this means is that if you do a double digest, you are likely to have linearized fragments that have been cut with only one of the enzymes. This would result in a hight level of religation.

hi,
i was having the same problem. first thing is check end-cutting efficiency of ur rest enz, could do it on neb site, then u can dephosphorylate ur vector and phos ur insert (in case, its not already phos, if u cut this also with res enz), use high ratios of insert/vector.
hope this helps.
good luck
nbj

Do you purify your double-digested plasmid on gel. You must, because even traces of remaining undigested plasmid would very efficiently produce unwanted colonies. Of course in blue/white screening that should not be a problem since those colonies would be blue.