fill-in remaining nicks in recombinant plasmid

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

Vagelis wrote:



Hmmm...is there a reason why you don't just do a prep from your
transformed E. coli and transfect with that?

Anyway...I think a solution would depend on the length and type of your
overhangs that you are using to insert your oligo...

cheers

Austin

I cannot use the plasmid from the transformed E.coli because a DNA
adduct is incorporated into the 114mer and the recombinant vector is
used for adduct site-specific studies.
The vector is digested with the XhoI and BamHI restriction enzymes
which both produce sticky ends:

XhoI
5'....C^TCGA G....3'
3'....G AGCT^C....5'

BamHI
5'....G^GATC C....3'
3'....C CTAG^G....5'


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