Hello, Sneha Paul,

I use 10 mM Tris, 1 mM EDTA and 20 mM KCl (pH = 7.5) for my spectroscopic experiments with G-quadruplexes (circular dichroism). Yes, many authors use cacodylate buffer, but I think that other buffers are OK too, the critical point for quadruplex formation/stabilisation is concentration of K+ ions or other additives (telomestatin, BG4, quadruplex binding proteins, etc.). But on the other side, most of buffers change their pH, if you increase/decrease temperature...Check this issue before starting your temperature dependent experiments.

Good luck !

Since ionic strength affects the melting temperature of DNA, you also have to consider the ionic strength of the buffer as a variable in the experiment.

Dear Sneha Paul,

To answer your question I have transcribed a paragraph from a very nice paper by Jean-Louis Mergny and Laurent Lacroix ( doi:10.1093/nar/gkw006) :

"For the melting experiments described in this protocol, using cacodylate buffer (pKa= 6.14) as the reference buffer for melting experiments is strongly recommended. It is important to choose a buffer that (1) does not absorb light in the far UV-region, (2) has a pKa close to the desired pH, (3) is relatively temperature-independent (phosphate and PIPES are commonly used for this reason), and (4) does not interact with other components of the solution (e.g., phosphate buffer should be avoided if working inthe presence of Mg2+or Ca2+). When an acidic pH is desired, acetate (pKa= 4.62) is acceptable. One should be aware that the pKa of other buffers such as MES, BES, TES,Tris, Tricine, HEPES, MOPS, and TAPS change significantly with temperature (in the range of 2 pH units between 0◦and 100◦C; Fukada and Takahashi, 1998). Therefore, these buffers are not appropriate for Tm experiments, even if the process studied is not pH dependent, as pH changes will also affect ionic strength of the buffer."

Moreover, by using lithium cacodylate which has little or no effect on G4 stability, (since lithium is considered "neutral") you are able to better modulate the ionic conditions of your buffer and FRET experiment.

Hope this helps,

Josué

Thanks Josue Carvalho for your reply. This answers my question appropriately. Since, I am using one of the more stable G-Quadruplex forming sequences, I believe phosphate buffer should not be a problem as you correctly mentioned that the pH does not change much with temperature and has pKa close to my desired pH of 7.4