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-   -   Annealing ssDNA oligos (http://www.molecularstation.com/forum/molecular-biology-techniques/85355-annealing-ssdna-oligos.html)

gmehta1 05-12-2012 10:10 PM

Annealing ssDNA oligos
 
Hello,

I have been trying for WEEKS to clone some short (30-50bp) complementary oligos into a 6kb double-cut vector. I've added the necessary sticky end overhangs to the oligos, but never any colonies aside from very occasional colonies without insert.

I've not tried dephosphorylating vector or phosphorylating oligos, because they are directional sticky ends.

I did the annealing in kinase buffer or ligase buffer, with v slow cooling rom 954 degrees. I've run the annealed oligos on a PAGE gel, and there are usually two bands with some smearing.

I'm really not sure what else to do, and its literally driving me crazy!! Any ideas would be very welcome!


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