We've had good success using the well solution directly as the foundation of a cryobuffer in several situations where crystals cannot be grown directly in the presence of cryoprotectant, and where crystals don't tolerate transfer to artificial mother liquors. The basic protocol is as follows:

1. Remove 100 microliters of the well solution after crystals have grown

2. Split this sample into two 50 microliter aliquots.

3. Add 7.5 mg of dextrose (glucose) to the first aliquot and 15 mg of dextrose to the second. Dissolve by gentle pipeting with a wide-bore tip. This will give two sequential well solutions that now contain 15% and 30% w/v dextrose. If all the dextrose won't go into the second aliquot, spin hard and remove the supernatant.

4. Transfer the crystal to aliquot 1, equilibrate for 3 minutes, then to aliquot number 2, then freeze. We've had a few crystals that routinely crack or blow up when transfered to artificial mother liquor that behave well when transfered to well solution plus glucose. We assume that there is some aspect of the crystal drop (pH, ionic tension, precipitant concentration) that is more effectively reproduced within the well than by separately prepared mother liquors. The nice thing about the protocol above is that you don't get much of a volume increase when dry dextrose is dissolved in the well solution, so the components in the solution are not diluted.

Finally, if you don't get a really good freeze, you can try to add about 5% v/v glycerol to aliquot 2 in addition to the 30% w/v dextrose. Reference: Personal communication from Barry Stoddard, Fred Hutchinson Cancer Research Center