fill-in remaining nicks in recombinant plasmid

Vagelis el36 at le.ac.uk
Mon Apr 25 12:00:42 EST 2005


Hello everyone, 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 with my vector, the
cells somehow (exonucleases?) chew my oligo, as the sequence data
suggest.
Do you know why this happens and how I can avoid it?
Is there any way I can seal the remaining nicks before transfection
into the human cells?
How about Taq polymerase, would it seal the nicks?
Any suggestions are welcome,
Thank you in advance,
Vagelis, Leicester



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