I have done digestion of vector pET21A with EcoRI and am seeing higher mobility of uncut versus cut vector? What could be the reason? Could I still go ahead with the cloning if the linearised size? Is that OK?
As you see in the figure Lane 1 is the uncut and the others are single digests of EcoRI and Xho1. I can't explain this.
Had such a problem earlier with pRSET A vector too.
Hi there,
Linearized vector runs less than circular supercoiled plasmid because the second is much more compact. So linearization of vector always result in increasing apparent size of plasmid. If you want to calculate size of a plasmid on agarose gel, linearize it first because DNA ladder are made of linear DNA.
Yes Dominque
The linearised band matches with the expected size 5.4 kb-the ladder is Lambda Hind III and my linearised band is between 6 and 4 kb so the size is OK but the uncut is running higher as you see in the gel-I read on a web site that this is due to nuclease contamination in prep or "harsh" purification
So is the non digested running faster or slower? Can you tell exactly who is who on your gel from left to right?
This is rather a very blurry image. When you run a circular vector then it normal runs faster than the linear one. And you also see different conformations of the circular plasmid. totally second Mr ligers' answer.
If you see the file below, you would found that undigested plasmid on the rightmost lane is runnig a little faster and also showing different conformations also
I ll relabel the lanes
Lane 1 :Uncut vector
Lane 2:Cut vector
Lane 3:Lambda Hind III digest
Lane 4:Cut vector
Lane 5:Cut vector
Hi again,
I have used pET vectors for years and I have never been confronted to this situation where it seems you haven't any supercoiled form of the plasmid in your prep (which is usually the prevalent form)... It may be your vector is nicked which results in relaxed form which is less mobile in agarose gel...
Yes I too have worked with pET and many vectors and this is the first for me too-in fact the whole of this year i ve been working with pET batches of plasmid and this is a recent develoment-would you still advise me to ahead for cloning if the linearised size matches with the expected 5.4 kb?
that was the advice I got on this forum a year back with another vector pRSETA which had a similar issue-please advise
Hi again,
If the problem is just nicked strand, it doesn't matter as the nick will be repair after transformation (the only risk is the nicked site being very close to the digestion site) : you can use the linear form as vector for ligation. The other solution would be to make a new prep of the plasmid and to see what happens on gel...
If I were you, I would go back to plasmid prep to get rid of any doubt. And check plasmid behavior on gel first and then perform digest.
Yes Dominique-I agree - I too have some old preps -can try out digestion patterns and then see if that is ok and then do ligation-thanks a ton for the advice-i will keep you posted.
Dominique _ I went ahead even with the funny mobility of uncut and got the clones -thanks for yr advice though -
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