Le Mardi 7 Juillet 2015 14:03 CEST, Almudena Ponce Salvatierra <maps.fa...@gmail.com> a écrit:
Hello Do you have full complementarity of DNA and RNA ? Does the difference in lenght (17 nt/19 nt) mean that you have a bulge on the DNA when the duplex is formed ? In short, what is the Tm of this duplex at these strand and salt concentrations? If the Tm is 35 °C, then "room temperature" may be a bit hot depending on where you are in this month of July. Also, what was the temperature of the gel during migration ? Do you have access to ITC ? If yes, we have found that this is a perfect technique (1) to check that you have formation of the duplex (basepair formation generates a lot of heat), (2) that you are using 100 % of each strand (or may be less...), (3) that you can stop injecting the second strand when the stoichiomertic ratio is 1/1 (no strand in excess: perfect for crystallization) and, finally, (4) that you can retrieve the sample from the cell, concentrate it and make crystallization drops. See: Da Veiga C., Mezher J., Dumas P., and Eric Ennifar (2015).Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes. In Nucleic Acid Crystallography : Methods and Protocols. (Ennifar E., ed..) in press. Humana Press. NY. Abstract I hope this can be useful. Philippe Dumas > > 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
