Annealing palindromic linkers

Bruno Cenni bruno.removethis.cenni at removethis.insel.ch
Wed Jan 17 11:16:26 EST 2001


We tried both to phosphorylate the oligo and not to...

Bruno


"Peter Ashby" <p.r.ashby at dundee.MAPS.ac.uk> wrote in message
news:p.r.ashby-8C5ED0.15571816012001 at dux.dundee.ac.uk...
> In article <3a63fb84$1 at news.unibe.ch>, "Bruno Cenni"
> <bruno.removethis.cenni at removethis.insel.ch> wrote:
>
> > Hi all,
> >
> > we're (expectedly) having troubles cloning a linker into a plasmid. This
> > linker consists of two complementary 33 bp oligos that after annealing
> > have
> > open restriction enzyme sites at their ends. The problems comes probably
> > from the fact that the oligos contain two complementary inverted 6 bp
> > recognition sites thus giving a palindrome (spaced by 3 bp). Any
> > suggestions
> > on conditions to increase the proper annealing of the oligos without too
> > much of the self-annealing and stem-loop formation?
>
> Are your oligos phosphorylated? it makes quite a difference when doing
> this and I would suspect that before assuming your oligos haven't
> annealed. A cheap way to achieve this is to make your oligos overlapping
> and then cut them after annealing.
>
> Peter
>
> --
> Peter Ashby
> Wellcome Trust Biocentre
> University of Dundee
> Dundee, Scotland
> Reverse the spam and remove to email me.







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