Hello.
I was looking back at some gel pictures of mine (because downstream work is not working...) and realized the electrophoresis for plasmid DNA is kind of odd.
From what I know, when plasmid DNA is run on an agarose gel, there are three bands: circular(or nicked), linear, supercoiled.
The picture I have included shows plasmid DNA in the 2nd lane with three bands but the bottom band aligns with single-site restriction digested results (all the other lanes to the right).
I've sent the same plasmid (from the same miniprep) for sequencing and I know the total size of the plasmid is ~4000bp, and this agrees with single restriction results. However, somehow when the plasmid is run on gel without restriction, there seems to be only linearized DNA.
So my question is, can there be no supercoiled plasmid after miniprep?
Any help would be appreciated.
Yes if the extraction method is harsh it might just create relaxed plasmid which looks like linear.
Jean-Pierre Dangy Thanks for the reply.
However, I'm following a simple routine miniprep protocol with a kit.
Can this be considered "harsh"?
Hello
After a miniprep by using a commercial kit, you are supposed to get supercoiled plasmid. At times, you might get three bands namely, supercoiled, linear, and nicked plasmid depending on the kit and the handling procedures.
In this case, you could consider the possibility of contamination of the resuspension buffer or the loading buffer with nucleases like endonuclease.
Repeated freeze-thaw cycles should also be considered.
Best.
Malcolm Nobre Thanks for your reply.
I understand that a miniprep with a commercial kit will result in supercoiled plasmids. I expect exactly that.
However, my gel seems to show three bands without supercoiled form.
Yet, when the same plasmid is restricted with a single restriction enzyme, I get a single band at the expected size.
So even if things were contaminated, there must be the full plasmid I was aiming to get in the first place.
The bands from restriction reaction show that I have the plasmid I want, yet the unrestricted plasmid seems to say otherwise. I don't think I'm getting a single band by chance because all the bands on the right are done with different restriction enzymes (XbaI, NdeI, and so on).
What could be the reason for seemingly opposite results?
If you are using a Kit then something is wrong, either you have vortex the denaturation mix or something similar because most of the kits should give you a vast proportion of supercoiled plasmid. As for contamination with endonucleases suggested by Malcom I don't believe it too much since ther's no really signs of degraded products on your agarose gel. Endonucleases usually degrade DNA down to nucleotides so you would see a huge bulb of small molecular weight products on your gel
Jean-Pierre Dangy Well now that is worrying... I still get the size I want with a single-cut reaction. I wonder what could be causing this
Well it's known that if you nick the DNA you will get a minority of supercoiled plasmid that doesn't mean degradation it just loose his highly coiled structure but most of the time the DNA is still usable for restriction digest, ligation and many other applications. The Supercoiled plasmid are usually essential for application like transformation of E.coli. Try to be gentle during the lysis process just invert the tubes several times to get an homogenous mixing but no vortexing and no brute force shaking. Also make sure that the denaturation step (NaOH+SDS) is not too long 2 to 3 mn is usually enough to denature chromosomal DNA and then immediately add the Solution 3 (KoAc) and invert several times but gently that should maitain a good supercoiled ratio
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