Hi Weifei, so... what I do is to mix them in a 1:1ish ratio, then I check that ratio on the HPLC in case I can adjust it to 1:1. I.e. Instead of mixing 30 nmol of RNA with 30 of DNA if I want a 1:1 ratio complex, I mix 30 of RNA and 25 of DNA. I calculate their molar extinction coefficient (epsilon) and check what is the ratio between the one of RNA and the one of DNA. Out of the HPLC I calculate the area of the peaks, divide one by the other one and check whether this ratio matches the one between the epsilons. If it doesn't then you calculate how much you still need to add to achieve this 1:1 ratio.
Depending on your desired final concentrartion you probably have to concentrate this RNA-DNA mixture before adding the buffer. I concentrate in viva spin colum and then add the buffer (mine is 10mM HEPES pH 8.0, 150 mM NaCl, and 2mM KCl. I anneal it at 95° for 3 minutes and then let it cool to room temperature for about 20'. After it cools down I add 20 mM MgCl2. I'm not sure this helps a lot or not... this is what I do... I don't know if it gives you any idea of what can be not working in your case. Good luck. Best, ALmudena 2015-07-03 17:14 GMT+02:00 ChenWeiFei <weife...@outlook.com>: > 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 -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
