I would like to inhibit the activity of two miRNAs, mir21 and mir133, using Tough decoy method. For this, I have designed some sequences and ordered those oligos, as you can see in attached file. In brief, the sense and antisense strands of each fragment (stem, loop and miRNA binding site) were prepared and ligated together. Then each decoy was ligated into the vector in the ratio of 1:3 and 1:7. I did cloning then, but I didn’t get any clone. I think the problem is related to the annealing and ligation of those fragments since they are short in length and there is a chance for miss-annealing. I would like to know how I should anneal those small fragments as a tough decoy construct and insert into the vector?
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