Cloning Double-Standed Oligos

Alexander N. Kukushkin kan at
Fri Dec 20 12:30:58 EST 1996

>   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

Hi, Lourdes
I have often performed ligation of ds oligos into the reporter plasmids. After
synthesis my single stranded oligos had 5'-OH ends and they were not
phosphorylated then. To anneal two primers the mixture was incubated at 85 C
for 10 min and further cooled slowly to room temperature. The buffer for
annealing contained 20 mM HEPES, pH7.7, 50 mM NaCl, 6 mM MgCl2.
For ligation I used Promega kit and vector/oligo ratio = 1/10. The ligation
continued overnight at 16 C. Usually I needed one copy insert of ds oligo
and got it well.
Good luck

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