Le Mardi 7 Juillet 2015 14:03 CEST, Almudena Ponce Salvatierra <[log in to unmask]> 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 <[log in to unmask]>:
>
> > 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
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