Can you give more details about restriction digestion and klenow treatment?

Hi Favio,

I am not quite sure, whether I understood you right but I assume that you ligate a double stranded oligo. Then I guess your problem is the oligo. My experience is that ds hairpin oligos are very unstable. I recommend an annealing buffer with at least 100 mM NaCl. Directly add your freshly annealed oligo to the ligation. Avoid prior dilution of the oligo in low salt buffers like TE.

Thank you very much for your comments.

I anneal the oligos in a NEBuffer 3 of New England Biolabs which has at least 100 mM of NaCl as Uwe says. Is strictly recommended the fresh condition of the oligos solution? Because we are now using a solution of more than 2 weeks. We mix 1 ul of 10 uM oligos solution, about 20 ng of digested plasmid and the recommended amounts of enzyme (or a little bit more) and buffer. What do you think about plasmid:oligo ratio? Many people suggest more oligos than plasmid but others use more plasmid quantity. Again thank you very much.

Sorry, I just now see your answer. In case of hairpin oligos I would use freshly annealed oligos, just to be on the safe side.

We use the following mix to anneal the oligos:

60μl TEN (40 mM Tris-HCl pH7.5, 1mM EDTA, 150 mM NaCl)

20 μl Oligo upper (100 pMol/μl)

20 μl Oligo lower (100 pMol/μl)

We add ca 0.1µl of the oligos to 10-20 ng of the vector. This is a still a huge excess of oligos. Since they are not phosphorylated, they don't form multimers. The vector needs the 5'-phosphate groups. Too much of the oligos inhibits the reaction.

I don't see why NEB3 should not work. If you are still not successful you may switch to a strategy without a Klenow reaction. At least one step less where something cango wrong.