how to distinguish supercoiled DNA from linear one in agarose gel - (Oct/07/2008 )
i would appreciate if any body could tell me how to distinguish supercoiled DNA FROM linear one by agarose gel electrophoresis!
i have actually searched on the web and found an article which is mentioned that we can distinguish them by gradient agarose gel electrophoresis but i was wondering if there is a simpler way! HOW LAZY I AM!!!!!!
here is the link for the paper i talked about
If you talk about plasmids then you can distinguish them with a regular Agarose gel. However, you need a control. For example supercoiled plasmid A with linear plasmid A. In this case, the supercoiled Version will run a little bit faster and will appear a little bit smaller then the linear Version.
Yes there is a simple way. You just need to set up two lanes. Lane 1 contains uncut DNA and lane 2 contains linearised DNA. Direct comparison of the lanes will tell you which species are relaxed nicked (upper band), linearised (middle band) and supercoiled (lower band). To make lane 1 as controlled as possible, include the same reactants as you do in your digest (lane 2), except for the restriction enzyme. Another piece of advice: the secret to easily distinguishing between the bands is to use the correct amount of DNA and electrophorese well. Too much DNA and a short electrophoresis will tend to cause the bands to bunch up and make them more difficult to distinguish from one another. If you really want to distinguish between the bands, set up a few controls and digests with different amounts of DNA to get the best picture. Using just one amount of DNA can sometimes be a stab in the dark getting the right amount of DNA, but if you are going to use just one amount, maybe 100 ng or so would be about right.