I am having trouble understanding what is the source of band "2" in the following gel-electrophoresis:


In this experiment, we took an E.coli transformant colony and ran its nucleic acids in the gel, after adding a restriction enzyme which cuts the recombinant plasmid molecule into two corresponding bands ("3" and "5" ["5" is very weak in the scanned version, but is well seen in the "hard copy" ) . I know the first line ("1") corresponds to possible genomic DNA we took from untransformed bacteria, and that the fourth line ("4") corresponds to a plasmid molecule without an insert. 6 is also well understood.

My question is- What is the source of band "2" which contains more base pairs than any of the parts of the plasmid and its insert , and in particular more base pairs than the insert?

WIll someone please help me understand this ?

Thanks in advance

  • $\begingroup$ I think you need some controls. If you can purify the plasmid away from the genomic DNA, run that plasmid alongside this lane. Would also be good to have genomic DNA without plasmid, such as from your untransformed control, in another lane. This should tell you if the mystery band is from the plasmid or the genome. If it's plasmid DNA, are you sure you're getting complete digestion? Uncut plasmids usually produce more than 1 band because supercoiled DNA migrates at a different rate than open circle, which is different from linear. Incomplete digestion could have left supercoiled and circular. $\endgroup$
    – user137
    Commented Sep 17, 2014 at 16:54
  • $\begingroup$ Hi user137, thanks for your answer, as for your suggestions: 1. I ran four transformed colonies in four different lanes, and this is the only lane when this mysterious line appears, so I guess the problem is not incomplete digestion. 2. I currently have no possibility of redoing this experiment with other controls. I am sure about the bands I told about, but still have no idea about this one. As mentioned here: biology.stackexchange.com/questions/19114/… the circular form should run a little bit $\endgroup$
    – joshua
    Commented Sep 17, 2014 at 17:01
  • $\begingroup$ less fast than the linear one, so I don't think it is reasonable that this line corresponds to a circular plasmid, since "4" is the line corresponding to a linear plasmid without an insert. WIll you please help me figure it out ? Thanks a lot ! $\endgroup$
    – joshua
    Commented Sep 17, 2014 at 17:02
  • $\begingroup$ So you combined different plasmid digests with genomic DNA from different samples of bacteria and ran it all in 1 lane? Please describe the digestion in more detail. Band 4 is empty vector, but what are bands 3 and 5? $\endgroup$
    – user137
    Commented Sep 17, 2014 at 17:10
  • $\begingroup$ I would also suspect single cut DNA here. Can you give us some details on the digest? $\endgroup$
    – Chris
    Commented Sep 17, 2014 at 17:22


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