plasmid excision problem

vagos el36 at le.ac.uk
Tue Aug 9 11:00:07 EST 2005


Hello, my name is Evagelos Liapis and I am doing my pHD in the
Department of Biochemistry, University of Leicester.

I would appreciate some help on this one:

I am working with a 5kb plasmid from which I remove a 114bp oligo and
replace it with a modified 114bp oligo. The vector is dephosphorylated
prior to ligation with the phosphorylated insert and thus my
recombinant vector contains two remaining nicks.

Now, when I transform E.coli with my recombinant vector the two nicks
are repaired inside the bacteria, as is evident from the sequences I
get back from the colonies, which suggest that the modified oligo is
successfully incorporated.
However, when I transfect Ad293 human kidney cells or human fibroblast
cells with my vector, the
cells somehow chew my 114bp oligo, as the sequence data suggest.

Initially I thought that there might be a methylation problem, since my
114mer oligo is synthetic and thus unmethylated. However, in vitro
methylation of the recombinant vector with dam and/or CpG methylases
had no effect at all.

Do you have any ideas why this might happen and how I can avoid it?
Could the fact that my vector is a relaxed circle be responsible for
this oligo excision? Have you heard before about similar problems with
relaxed plasmids?

Any ideas are welcome.
Thank you in advance,
Vagelis, Leicester



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