Question on plasmids - (Jul/25/2015 )

Dear all,

I ran some plasmids on a gel and I see more than 1 band in some of the samples.

Now it should be 1 plasmid and I am aware that there are different types of the same plasmid (supercoiled, linear...) however how big could the difference be between them? Is it possible they differ on a gel in terms of 1000-4000 bps? 

Or could it be I see different plasmids, some sort of contamination?

Yes, the different forms typically show a range of sizes. Only the linear runs as expected.

It could be completely normal that you have these different forms of the one single uncontaminated plasmid. Large differences in size shouldn't be surprising. To check for contamination, restriction digest to linearize the plasmid and then gel migrate.

bob1 on Sun Jul 26 08:14:21 2015 said:

Yes, the different forms typically show a range of sizes. Only the linear runs as expected.

It seems weird they can differ so much in size on a gel.

gvbdxz on Mon Jul 27 10:16:47 2015 said:

It could be completely normal that you have these different forms of the one single uncontaminated plasmid. Large differences in size shouldn't be surprising. To check for contamination, restriction digest to linearize the plasmid and then gel migrate.

It looks like that I have 2 plasmids in a few bands since the digest I did gave 2 bands while it should just be 1.

Unless some uncut DNA stays on top? But how do I now this is the case? 

So hard to tell from a gel!

It's not weird how they migrate so differently, it is all about the conformation of the DNA and how it passes through the pores in the gel. 

It is entirely possible to get undigested plasmid in your sample, if you really want to know, digest with a RE that will result in multiple bands. The banding pattern should vary between plasmids of different backbones, and you can use a number of tools to give you virtual gels to compare to (enzymeX is a good one, but there are heaps of others).  If you are still concerned, then you could sequence over the insert site, and this should tell you if you have more than one plasmid present.