I tried to insert a 6.6kbp gene into a 3.8kbp using CloneEZ, and transformed it into E. coli. After that a colony was selected and grown in 10mL of media for miniprep.
I did a gel run on the plasmid freshly purified from the grown bacteria, but upon my gel run I found that the bands were slightly below 3kbp, which was even smaller than the empty vector itself.
My question is thus this: is it possible that a supercoiled 10kbp plasmid run all the way down to 3kbp? Or should I assume that that band is actually 3.8kbp plasmid, i.e. my gene did not insert?
Hi,
it is hard to say. Supercoiled plasmids are unpredictable beasts - you never know, how fast they can be during elfo. I would not exclude the possibility, you actually have the desired construct. To be sure, linearize it as Dereck suggested or run PCR - insert specific; vector specific (with primers spanning the cloning site) or vector -insert specific (one primer in the insert, the other in teh vector).
Hi Jackie,
I am assuming that you are using a molecular weight standard comprising linear fragments. If so, then it is not possible to accurately size a supercoiled plasmid as there are many factors that would need to be taken into account (type of agarose, gel strength, buffer composition and concentration.
You should either run your supercoiled plasmid DNA on a gel using a supercoiled molecular weight marker (I use one from New England Biolabs which contains ccc plasmid moleculaes from 2 up to 10kbp), or linearize your plasmid with a restriction enzyme to run with your linear molecular weight marker.
I hope this helps.
Hi,
it is hard to say. Supercoiled plasmids are unpredictable beasts - you never know, how fast they can be during elfo. I would not exclude the possibility, you actually have the desired construct. To be sure, linearize it as Dereck suggested or run PCR - insert specific; vector specific (with primers spanning the cloning site) or vector -insert specific (one primer in the insert, the other in teh vector).
Hi Jackie,
Yes it is possible that the supercoiled 10kbp plasmid runs down to linearized 3kbp. In your case, just digest the plasmid to see its actual size.
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