I am working with large plasmids, ~20kb. I am using an alkaline lysis method for extraction, however I am not sure that it is pure. I see two sharp bands at the top of the gel, which I assume to be relaxed circle and linear bands, however I also see the majority of the stained DNA in the ~1kb region. I am trying to decide if this is my supercoil, or something else. I am not sure how much faster supercoil will run in a gel relative to the linear forms.
Firstly, as mentioned above, for a 20kb plasmid it is unlikely to see the supercoiled form at 1kb.
Supercoiled form runs faster than linear plasmıd DNA and you can check the relative positions of different plasmid DNA forms from this link: http://hg.wustl.edu/hdk_lab_manual/image/plsmid3.gif
Supercoiled DNA band appears crescent-shaped. This may also help you to identify it.
If your vector is around 20kb then I wouldn't expect the supercoiled version around 1kb. This is either an artefact from your clean-up or your vector is broken.
Firstly, as mentioned above, for a 20kb plasmid it is unlikely to see the supercoiled form at 1kb.
Supercoiled form runs faster than linear plasmıd DNA and you can check the relative positions of different plasmid DNA forms from this link: http://hg.wustl.edu/hdk_lab_manual/image/plsmid3.gif
Supercoiled DNA band appears crescent-shaped. This may also help you to identify it.
When we isolate plasmid and check on the gel. Normally two bands can be seen on the gel. The top one band is of nick circular DNA. It does not move too for. and the lower one is covalently closed circular DNA. Bands of linear plasmid you will never get untill or unless you do double digestion of Plasmid.
Dear Nat, the mobility of your plasmid cannot be close to mobility of 1kb. Please, verify whether your RNase still works properly during isolation. Usually at this position is observed RNAs. Another possibility is following. If you leave your sample od plasmid 1-5 days at room temperature, RNAs are digested, DNA is stable, afterwards try to load the sample and do electrophoretic separation, in case 1kb band will partially/totally disapear then you had observed RNAs.
This answer is for regular mini gels (not pulsed field).
For small plasmids, SC always runs faster, then linear, then open circular However, at some point, (20 kb or so, not sure about exact size), the supercoiled will migrate equally fast as the linear, and for large plasmids, supercoiled will run slower than linear but faster than open circular. Actually, at that point, linearized will all run the same since it can not be resolved on a minigel. That is why you can size a 30 kb, 50 kb, 70 kb and a 90 kb, 110 kb plasmid based on supercoiled and not linear form on a minigel, with an appropriate marker such as supercoiled DNA marker from Epicentre. The linear form for all 5 plasmids will run at the same height on regular minigel. If you want to size based on linearized you will have to run a pulse field gel for such a plasmid.
Nat if we go for isolation of large size plasmids (Larger than 7kb) Than we mainly observe only two band in that case we wont be able to get separate band for open Circular and relaxed form so according to my experience i can say the two band you observed, one of them is super coiled form of plasmid.
The material you see at the 1 kb area is most likely RNA that did not get digested. Since you are working with a 20 kb plasmid, the two bands are most likely 1) nicked and supercoiled that migrate together 2) open circular. If there is band stuck in the well, that is open circular as well. This portion is trapped DNA. Some additional info can be found here (they also discuss regular gel separation of large plasmids):
Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning.
Wang M, Lai E.
Electrophoresis. 1995 Jan;16(1):1-7.
Nat, I agree with the others that the 1 kb is most likely RNA. Check out our results when using PEG and calcium chloride if you are trying to avoid RNase.
http://144.206.159.178/ft/2795/589011/12064016.pdf
In native gels run in Na acetate buffer supercoiled DNA runs to a position different than in Tris-borate buffer. One is faster than the other
You can run a "standard curve" with plasmids of known size also for supercoiled forms as an estimate of how big your plasmid is. Otherwise, I agree that about 1 kb linear is almost impossible for such a large plasmid. 2 comments extra: I remember a "poor man's pulse field GE" from the early 90s that used SDS ; another complication you might consider is that the yield of very large plasmids can be quite low and you may have to adjust your culture and extraction procedure. Good luck! WAS
Well, in o.4% agarose-thedium bromide gels you can get reliable MW distributions of ds fragments in the range 10-40kb
The three forms of an uncut plasmid, all of the same size and molecular weight, do not behave the same way during electrophoresis. Because the supercoiled molecules are more compact, they move more easily through the gel, and are found farther from the point of origin in the well. The relaxed circular molecules, and the full length linear molecules usually run very close together and are not always resolved from one another. When they do run as separate bands, either relaxed circles or linear may run faster in the gel, depending on whether the gel is run in the presence of ethidium bromide or not.
dear nat your plasmid size is much more larger,so some handling problem is their like it is not cut at proper position and you find some linear as well as few suppercoiled DNA.
if you are sure that the band of 1 kb is DNA, you can elute this band of 1kb and cut with restriction endonuclease which has a unique site (or more) in the plasmid. if the analyzes on electrophoretic gel, if it is the plasmid of 20kb should have the linear form (or have a number of bands corresponding to the number of cuts), otherwise it is not.
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