Another thing to try when cryoprotecting any crystals grown at room temp using a volatile precipitant is to first transfer the crystal tray to 4 degrees, then quickly add a cold aliquot of a viscous cryoprotectant to the drop. Evaporation is considerably slower at 4 degrees, and I prefer to use glycerol as cryoprotectant because it significantly slows down the "random walk" of the crystals through the drop. It makes it so much easier to fish crystals out of the drop if they're not moving at lightning speed...
Place the tray at 4 degrees about 20-30 minutes prior to opening the drop. Any longer incubation at lower temp can result in extra nucleation or crystal dissolution. BTW, good luck trying to use a really volatile organic as a cryoprotectant. Every few years I try it and the crystals just get blown out of the cryoloop by the nitrogen gas stream. Diana On Jan 24, 2014, at 5:31 PM, "Mark van der Woerd" <mjvdwo...@netscape.net<mailto:mjvdwo...@netscape.net>> wrote: Chen, Dioxane is not easy to work with, exactly for the reasons you describe. There is one thing you did not mention, which I know to be an additional issue: the quality of the dioxane. I do not know if you need good quality (whatever that is) but it is a fact that crystallization works with dioxane from some manufacturer/lots and not with others. I have never figured out why this is so. For a paper on kinetics and reservoir volume discussion, I would read the work by Forsythe et al (http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, doi:10.1107/S0907444902014208)<http://dx.doi.org/10.1107/S0907444902014208> Basically you will find that this paper says that well volume does not matter much. Content of your drop matters a lot. In your case, having a very volatile component, equilibration would be really fast. One way to get away from all that is to do batch experiments, where there is no active transport from a drop into a well. But your question remains very good: you need an "oil" (?) that will not permit water or a volatile organic substance to escape. I am not sure what would do the trick. Dioxane is a decent cryo-protectant by itself. I would think about adding "gooey" things like low molecular weight PEG to try to get the drops better behaved. Evaporation of the dioxane will still be an issue, but with a higher viscosity you may not have to "chase your crystals" through the drop. Hope this helps a little. Mark -----Original Message----- From: Chen Zhao <c.z...@yale.edu<mailto:c.z...@yale.edu>> To: CCP4BB <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Sent: Wed, Jan 22, 2014 11:00 pm Subject: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on "dioxane-impermeable" oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen ________________________________ UT Southwestern Medical Center The future of medicine, today.
