Cloning Double-Standed Oligos
seppen at u.washington.edu
Thu Dec 19 15:53:18 EST 1996
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.
> Lourdes Rodgers
> Graduate student
> The University of Michigan
> Ann Arbor MI
I just cloned a 34 bp site, this is how I did it. I made two complementary
oligo's, with some
overhang on each side so they would fit in a vector cut with the
complimentary sticky enzyme.
Combined both oligo's at high concentration in 1X SSC and boiled for 5
min. Slowly cooled to RT. Measure concentration and take a little bit for
Phosphorylation with T4 kinase. Ligate into vector. Works well. Be careful
that you do not get concatamers ligated. I sequenced several clones and 1
out of 4 had a tandem insert.
Hope this helps,
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