Hi Lingyuan I would certainly make a seed stock and use it with RANDOM screens - this will (usually) allow you to pick up new conditions and control the number of crystals per drop.
However you say your crystals melt easily when you harvest them. I don't know how David stabilized his seedstock (if he did) but you may be able to stabilize yours with a precipitant. In all the cases that we looked at we found that if you soaked uncrushed crystals in a precipitant (or cocktail) and the crystals were unchanged after 24 hours, then the precipitant/cocktail could be used to make a seedstock. In practice 100% PEG 600 worked for 5 of the 6 model proteins that we looked at. The remaining seedstock worked with seed crystals suspended in 4M amm. sulfate. I hope it works for you. Best wishes, Patrick ___________________________________ Ref for stabilizing seed stocks with various precipitants: Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock, P.F., 2011. Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization.*Crystal Growth & Design*, *11*(8), pp.3432-3441. Ref for random microseeding: D'Arcy A, Villard F, Marsh M. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica Section D: Biological Crystallography. 2007 Apr 1;63(4):550-4. On 19 October 2016 at 15:20, Hargreaves, David < [email protected]> wrote: > I have been presented with crystals grown in nmr tubes on two separate > occasions. Both diffracted very well and thankfully, neither required a > magnetic field for optimisation. On the first occasion I did use the > initial sample as a seed stock. > > > > Best wishes, > > > > David > > > > *From:* CCP4 bulletin board [mailto:[email protected]] *On Behalf Of * > kiki > *Sent:* 18 October 2016 11:42 > *To:* [email protected] > *Subject:* [ccp4bb] Crystals grow at the bottom of the tube. > > > > Dear all users, > > > > I got my crystals at the bottom of the tube on ice just before I started > to screen crystals. Those crystals' shapes were good but easy to melt when > I harvested them. I shot these crystals. They only diffracted to 7A to 8A. > Has anyone ever met this situation before? How to improve? > > > > Thanks, > > Lingyuan > ------------------------------ > > AstraZeneca UK Limited is a company incorporated in England and Wales with > registered number:03674842 and its registered office at 1 Francis Crick > Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0AA. > > This e-mail and its attachments are intended for the above named recipient > only and may contain confidential and privileged information. If they have > come to you in error, you must not copy or show them to anyone; instead, > please reply to this e-mail, highlighting the error to the sender and then > immediately delete the message. For information about how AstraZeneca UK > Limited and its affiliates may process information, personal data and > monitor communications, please see our privacy notice at > www.astrazeneca.com > -- [email protected] Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
