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Electrophoresis of a plasmid

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Electrophoresis of a plasmid

Postby ZaraR » Thu May 05, 2011 4:05 am

One of the questions that we were given for an assignment was to predict to be the # of bands that would form on an agarose gel after electrophoresis of a plasmid which contained one kanamycin gene (1875 bp) and one ampicillin gene (3755 bp), and to explain our prediction. We didn't really learn much about this in class; it's more of an individual learning assignment. My guess would be that there would be two bands formed on the agarose gel, since their are only 2 genes. Is this correct, or am I misunderstanding something?

Any help is very much appreciated!
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Postby JackBean » Thu May 05, 2011 5:12 am

The number of genes doesn't matter. If the plasmid is intact (not cut by any restriction endonuclease), you will get 2-3 bands usually. The reason is here:
http://en.wikipedia.org/wiki/Plasmid#Conformations
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby protold » Thu May 05, 2011 5:28 am

Hola, plasmid have not only resistence genes, they have other fragments to assure their replication. look in the net for any plasmid map as pUC 18 or pBR322 ones of the simplest .All the plasmid could have in a gel three bans depending of the quality of preparation. Upper is supelcoil band, medium linear plasmid (broken in two bands) and lowest nicked form where one of chains is broken. Unless you have two resistence genes in the same plasmid, it, the plasmid is one unit and could show the three bands above mentioned, two or one. Different case is if you digest it with a RE wich have only a site of cut you will see only a band,: linear digested , if you cut at same time with RE which only cut on the sides of resistence genes you will have one band for any of them and the rest, thinking in that this RE don´t cut in any part of rest of plasmid. But I repeat without digest you have only the typical three band mentioned above. Buena suerte
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