Hi,
I am trying to get plasmid profiles from clinical isolates. I extracted them and visualized them in the gel electrophoresis. I tried to cut them with a 6bp enzyme XbaI to have more ideas on the size of the plasmids (anyway, I chose an enzyme randomly, since I don't know the sequence of the plasmid). I have some difficulties in the interpretation of the data and I attached the file (sorry the image is not great). They are 6 isolates and for each I run the cut and the uncut plasmid. I don't know how to determine the size of the plasmid and how to determine if I have more plasmids in my extract.. For example, as to the first isolate (K1), the cut produces 2 bands, one of which is larger than the only one band visible in the uncut product.. in your opinion, could the bands be the two pieces after cut of the plasmid? If yes, why are they larger than the single band visible for the uncut plasmid? does it mean that the uncut product is a supercoiled form e migrate faster?...the other example, the cut of the plasmid extract of K5 produces different bands, again, larger than the K5 uncut. Are they pieces of the same plasmid after cut or different linearized plasmids?Again, the fact that I have bands larger for the cut plasmid DNA can suggest that the uncut plasmid is in the supercoiled form? I used the 1kb ladder
supercoiled plasmid dna runs quicker ( smaller) than open circle or cut linearised or nicked plasmid dna as the supercoiled form is knotted and actually forms a smaller molecule than the other type. A good gel picture is on page 1 of the attached file
I agree with David's answer which is a better answer to the question you posed.
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