The concentration of oligonucleotide seems much lower than you will ultimately need for biophysical work. Annealing two strands is a bimolecular reaction and it will be driven by mass action more to completion at higher total oligonucleotide concentrations. I also agree with the posts that have suggested higher salt concentrations. The charge density of double-stranded nucleic acids is much higher than for extended or even helical single-stranded nucleic acids. Higher salt will more effectively screen those charges and promote double helix formation. Tris would not be my first choice for pH control in a sample that is going to experience a 75C shift in temperature. The pKa temperature coefficient for Tris is about -0.03 pH units per degree C, so the pH will change by over two pH units during your annealing protocol. If you are starting at pH 7.5, and 95C the pH will shift to just above 5. The pKa of cytosine is about 4.5 in solution, and in a highly negatively charged oligomer, it should shift to higher values. Protonated bases will not form Watson-Crick base-pairs as expected. I think you might do better with a buffer with a lower temperature coefficient. Back in the day, we used to anneal our oligonucleotides in cacodylate buffer. Sure it is toxic but it has a nearly flat temperature dependence and very few bacteria will live on cacodylate.
On Jul 3, 2015, at 10:14 AM, ChenWeiFei <weife...@outlook.com> wrote: > Dear all, > I want to get a complex of DNA-RNA-protein. But I have a big problem of > annealing DNA-RNA. > The length of DNA is 19nt and RNA is 17nt. > Annealing protocol: > 2uM DNA > 2uM RNA > 10mM Tris-Hcl > 100mMNaCl > 1mMEDTA > > Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature. > > I can just get a result of two single strand DNA/RNA. PAGE analysis. > > No double helix was founded. > > Does anyone have the same problem or know how to fix it. > > Thank you for your answering. > > Best regards, > Weifei
