Cloning Double-Standed Oligos

Jeff "Newt" Sekelsky fznewt at peseta.ucdavis.edu
Fri Dec 20 14:58:11 EST 1996


I often do this, and I don't phosphorylate.  The advantage of not kinasing
the oligos is that you won't get concatamers.  The disadvantage is that
you can get recircularization of the vector.  Two ways to prevent that: 
1)  directional cloning; 2) design the oligos such that the cut site is
destroyed if the oligos ligate in, then cut the ligation with the original
enzyme before transformation.

One useful trick I just learned when using non-phosphorylated oligos: 
Ligate with an excess of annealed oligos, then heat the reaction to 80 C
(or so), spin through a Microcon (or similar) to remove unligated oligos,
and allow the remainder to reanneal at low concentration.  When the
annealed oligos are in excess, a common ligation product is one with a
different ds oligo at each end of the vector (especially if the vector is
large).  The free ends can't ligate to one another because neither has
PO4.  The vector-oligo junctions are nicked due to the non-PO4 oligo ends,
so one oligo strand is removed by the heat.  The remaining oligo ends can
anneal to one another, making a nicked ds circle, which will transform.

JS


On Thu, 19 Dec 1996, Lourdes Rodgers wrote:

> If anyone has experience with cloning double stranded oligos please send 
> me useful tips such as: should the primers be phosphorylated?, tips on 
> annealing primers, ligation reaction conditions, etc. 
> Thanks,
> 
> Lourdes Rodgers
> Graduate student
> The University of Michigan
> Ann Arbor MI
> 
> 




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