I want to anneal 2 oligos but they are forming a hairpin. I will denature them at 95 degrees, but will the hairpins form again as I lower it to room temp?

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Barbara Rieck

How much do these oligoes overlap? How do you know if they form a hairpin - by actual experiments or by in silico analysis? I would try to denature them and lower the temp slowly. If that does not work, I once annealed oligoes by using PCR and I was able to see my fragment in a gel later confirming that they annealed.

Reema Dhoke

Thanks Barbara, haven't got my oligos yet, had just done in silico analysis and found them to form loops with Tm value for forward as 32 and for reverse as 73. I have decided to denature them to 95-100 degrees and hopefully when I run the gel, they will anneal. When I do the experiment I will get back to you.

Thanks alot.