Plasmid DNA preparation - two DNA bands in gel (May/01/2008 )

Hi all,

I did miniprep using QIAGEN miniprep kit and i am seeing two bands when the plasmid DNA is run in agarose gel. The top band (around 9kb) is very thick and the lower one (around 8kb) is a thin band. I am expecting plasmid DNA of size 8kb...i am so confused..are both bands the same, one is supercoiled and so has run faster than the other ? can anyone explain why this is happening? thanks in advance

- Paapu

If plasmid is uncut, the supercoiled should run faster than the nicked/relaxed. The bulk of the DNA should be supercoiled, so I don't know why you see the opposite pattern. Could be two types of plasmid in prep (one without insert, or recombination occurred). You'll know the deal once you linearize it.
d

supercoiled plasmids have a highly variable running rate, sometimes they will run slower, sometimes faster, this is due to the conformation of the supercoils. If the coils are forming the DNA into a large lump-like mass then it will run slower, but if the coils form it into a smaller more compact mass, then it will run faster than the expected size.

BTW, you should have three bands on your gel - supercoiled, uncut and linear plasmid DNA.

I agree with Rose... usually the dominant form is supercoiled, which runs the fastest. You really shouldn't see too much linear in a plasmid prep unless the isolation conditions were particularly harsh. It sounds like you may indeed have two versions of the plasmid in your prep...

On second thought though, sometimes the plasmid is nicked during prep and you convert a lot of it to open circle -- in which case, it would run higher than the sc form... Hmm. Well, in any event, like Rose said, you'll know when you linearize everything.
Good luck.