Hi! Vacuum oil can effectively stop evaporation completely if immediately dispensed onto the drop after the cover slip is removed from the well. 2x-5x the drop volume dispensed directly onto the drop creates a "drop within a drop", where air is prevented from reaching the inner drop, but the outer oil drop and inner crystal drop do not mix.
This technique is limited however by its difficulty in harvesting crystals out of the crystal drop through the oil. Also, while the oil itself will freeze clear, the crystal may not be cryoprotected sufficiently. Crystals can be transferred by pipetting them out of the inner drop to a separate well for stepwise cryoprotection. As mentioned below, a 500uL well at 4 degrees C will evaporate much slower and lead to much less turbulence than a few microliters at 20 degrees C. Disclaimer: I was using crystals that never diffracted anyway so this technique's utility may be an urban legend :) -Aaron Brewster -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Charu Chaudhry Sent: Tuesday, August 26, 2008 9:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Handling protein crystals from 20% isopropanol Hello, My crystals in fact grew out of 20% peg4K and 20% isopropanol at 20 degrees and so were already effectively cryoprotected in the drop. Opening up the well at room temperature led to the crystals whirling around in the drop and made it hard to pick up a single crystal in the loop. The effect is not as bad at 4 degrees however and this has been other people's experience as well. By increasing the viscosity by adding peg to 30% instead of using higher concentrations of isopropanol as the cryoprotectant and moving the tray to 4 degrees, the solvent movement was slowed dramatically and I could work quickly through sequential transfers in 5% peg increments into my final cryoprotectant. Of course you need to check that there isn't a deleterious effect of moving your crystals to 4 degrees if they were not grown there. Hope this helps, Charu On 8/26/08 11:17 AM, "Narayanan Ramasubbu" <[EMAIL PROTECTED]> wrote: > Dear All: > I have obtained good crystals from 20% isopropanol but this is my first > time dealing with this agent. > Help in handling the crystals, dealing with evaporation etc. is highly > appreciated. > Thank you > Subbu
