Generally, 5 mins at 65 degrees is regarded as "sufficient to denature secondary structure", while also being "not long or hot enough to cause hydrolysis".

It's what most cDNA synthesis methods use as the initial denaturation (for annealing primers).

Hi there,

Heating at 65-70°C is OK as long as the pH remains acidic and there is no RNAse in the sample. Leaving the sample to cool down at room temperature is fine for renaturation.

Denaturation can be performed by adding urea (1M) but then renaturation requires urea to be removed/diluted...

Thanks for your helpful insights, John Hildyard and Dominique Liger