Ligase free annealing question

EK someone at microsoft.net
Fri Jan 23 12:32:35 EST 2004


Thanks Hiranya,
For annealing with the vector, I will not use ligase and rely solely on
annealing of complementary sticky ends on the vector and the inserts. The
reason for it is that I believe it will reduce the anount of self ligated
vector. I do get clones of self ligated vector if I use ligase, despite the
noncompatible ends.
Emir

""Dr. Hiranya S. Roychowdhury"" <hroychow at nmsu.edu> wrote in message
news:1074876628.401150d452856 at webmail.nmsu.edu...
> Emir,
>
> This is a very simple protocol that I use:
>
> Mix the complementary oligos in equimolar concentration (TE, pH7.5 or 8
with
> 50mM NaCl) in a 1.5mL tube.  I usually start with about 500picomoles of
each.
>
> Boil about 500-700 mL water in a 1L beaker, remove the beaker from heat
source
> (microwave or burner or hotplate), and float the tube of oligo mix on the
hot
> water (using a floatie, of course).  Let the water cool to below 30
degrees.
> When I do it, I simply let it sit on the benchtop overnight.
>
> Alternatively, you can also do it in a thermocycler using the preset
> "denaturing" (95 C) for 5min, and then programming the block to cool to 25
C
> over at least an hour.
>
> The anealed oligoes are stored frozen at -20.  In some cases, such
annealed
> oligoes may need to be re-annealed before using after a prolonged storage.
>
> You may use kinased oligos (or is it "oligoes"?) or you may kinase the
annealed
> duplex prior to ligation.
>
> Hope this helps.
>
> Hiranya.
>
>
> Quoting EK <nobody at elnino.com>:
>
> > I need to do the following:
> > 1) mix and anneal 2 primers that after annealing should produce a 36bp
> > double-strand region and 15nt sticky ends on both sides. This product
will
> > be used as an insert.
> > 2) use above insert to anneal with the linearized plasmid having
> > complementary sticky ends to the sticky ends in the insert.
> >
> > Does anybody have any suggestion on how to increase the yields of both
> > annealed inserts and efficiency of insert incorporation into the
plasmid?
> >
> > I know this works, but unfortunately I don't have a detailed protocol.
> > Important parameters like concentrations of oligos in the annealing
> > reaction, buffer and salt concentrations, temperatures, ratios of
> > molecules,
> > etc. are missing.
> >
> > Any reference or pointer would be highly appreciated.
> > Thanks.
> > Emir
> >





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