fill-in remaining nicks in recombinant plasmid

Vagelis el36 at le.ac.uk
Tue Apr 26 07:51:55 EST 2005


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'


"Austin P. So (Hae Jin)" <nobody at nowhere.com> wrote in message news:<d4jd1p$i8a$1 at nntp.itservices.ubc.ca>...
> Vagelis wrote:
> 
> > 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?
> 
> 
> 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



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