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Title: how do you determine the orientation of an EcoRI fragment that has ligated onto a plasmid vector? Post by: sm1tty42 on Sep 12, 2012 I was told that there are two possible orientations, but I don't see how this is possible, since the EcoRI cuts the plasmid only once, and the fragment (also cut with EcoRI) can only ligate to the plasmid in one way (true, it can ligate to itself and then ligate onto the plasmid, but we're just talking ligating to the plasmid itself here).
Title: how do you determine the orientation of an EcoRI fragment that has ligated onto a plasmid vector? Post by: !JRon@n on Sep 12, 2012 The EcoR1 restriction enzyme cleaves a palindromic sequence. This means that the sequences on either of the sticky ends are identical. The EcoR1 sticky end on the fragment can attach to either sticky end of the EcoR1 digested plasmid. You could sequence the DNA by using a different primer than EcoR1, but I don't know if that would work out because you would have a solution with both orientations. I don't think you can figure out the orientation of a plasmid in one specific plasmid.
Good luck! |