The vector became smaller - (Nov/09/2008 )

Hello everybody,

I ligated the gene of interest in my vector, the restriction enzyme cutting and sequencing results showed that the fregment is right. But it was surprising that the vector is smaller than the empty vector. I did it several times and gained the same result. But why?

Thanks very much for giving me any advice.

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Are you comparing the ligated closed vector+insert to a linear vector only? Closed/supercoiled pDNA runs faster than the linearized form, and will probably show a rather diffused band where the linear will be comparatively sharper.

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No. I used the same enzymes flanked the gene of interest to cut both the empty vector and the ligated one.

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I hope you mean you just used one of the enzymes flanking the GOI. If you used both, you would have cut the gene out, and you'd have 2 fragments: the excised GOI and the vector, less any DNA you lost when you did the original vector preparation. How big is the vector backbone and what's the distance between the enzyme sites you used for cloning?

Try cutting the backbone and your recombinant vector with one enzyme and then do the comparison.

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Presume that the distance between the two restriction enzyme sites you intended to use is not large, but maybe one of these has another site that you didn't know (I was in the same situation before, just because I didn't analyze the vector sequence carefully), then you would lose an unexpected fragment.

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