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Dear All, Here are the main suggestions I received. I'm going to try them all. Many many thanks for your help. Yours, Claudia Possible solutions: 1. Maybe adding the cryo to the crystallization already? 2. 2R3R-Butandiol is the best cryo as being used in very low amounts. 10% are typically enough. 3. Put a small droplet of paratone-N next to your drop such that they touch and then drag the crystal into the oil and take care to remove the water from around it (the buble aorund it within the oil) and freeze. When draggin into oil go loop first such that the loop plows the way a bit (or some part of it) mechanical sress can be a problem due to the viscosity, but otherwise it often works, though not always.. 4. 2M amm sulfate might be quite enough if you do the transfer and dipping quickly. 5. other option is to add salt. e.g LiSO4 should be good cryoprotectant (see cryosalts) at high conc. 6. Have you tried with ethylene glycol for example? 7. Maybe you do not need any cryoprotectant and crystals mountd in small loops will freeze just fine straight out of your drops... 8. Under what conditions did the crystals grow? If its sodium acetate and ammonium sulphate I would try adding sodium malonate as a cryo protectant (with 2-2.3M ammonium sulphate adding 0.6-0.8M Na malonate should be sufficient) or increasing the ammonium sulphate concentration. Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 From: [log in to unmask] To: [log in to unmask] Subject: Re: [ccp4bb] Freezing under oil Date: Thu, 4 Feb 2010 11:52:03 +0000 Could you try using ammonium sulfate as your cryoprotectant? What happens if you slowly increase the concentration over time to >3M, followed by flash freezing? What about other cryos, like ethylene glycol? lpj Lauren Jackson, PhD [log in to unmask] On 4 Feb 2010, at 09:54, Claudia Scotti wrote: Dear list, I'm trying to freeze crystals in cryoconditions containing the following: 0.1 M Sodium acetate pH 4.4 2.15-2.3 M Ammonium sulphate 7% n-butanol 15% glycerol The problem is that the crystals (beautiful hexagonal prisms) seem to shatter in a random fashion: some are unaffected, some, coming from the same drop, disgregate miserably. One even split into three parts, of which two disgregated and one survived perfectly well. I've tried both by moving the crystals directly in the cryocondition and by progressively increasing the glycerol concentration to no avail. Shall I just select those that survive? I was wondering if anybody has ever had this same problem and if freezing under oil could be an alternative. If yes: any suggestions on how to fish the crystals? I tried it in the past, but I found it very difficult... Experiences and suggestions are welcome. Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 Hotmail: Trusted email with powerful SPAM protection. Sign up now. _________________________________________________________________ Hotmail: Powerful Free email with security by Microsoft. https://signup.live.com/signup.aspx?id=60969