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Hello Maria, the success of this method depends on a few things. First, your seed stock-solution. Most people prepare it by smashing up seeds in mother liquor (i.e. reservoir solution). Looking at the phase diagram, you will quickly understand that this might not be the best way to go as you will dissolve your seeds fairly quickly over time. That is why people prepare really fresh stocks, keep them on ice and freeze them as soon as possible in order to "stabilize" the seeds. Keeping on ice will only help if your protein is less soluble at lower temperatures, which true for roughly 45% of proteins (45% will react retrograde, 10% do not show a clear preference). Looking at the phase diagram again, you will also understand that seeding only works for "near" equilibrated drops that are metastable-supersaturated. If undersaturated, seeding will not work as the seeds will dissolve over time. Keeping the above considerations in mind, best is to seed after the drops have equilibrated. Next best option is to add the seeds or the protein as the last component to the drop... Jeroen. On 20.09.10 09:53, Maria Håkansson wrote: > Dear all, > > Anyone who have had success using the mosquito for micromatrix seeding? > Which way is the optimal way of adding seed solution to sitting drops > on an MRC plate? > > Is it better to add 100 nl protein + 10 nl seed solution + 100 nl > reservoir solution or > to add concentrated seed solution (1 microliter) directly to the > reservoir solution and then pipett > 100 nl protein + 100 nl reservoir solution. > > Best regards and thanks in advance, > > Maria Håkansson > > >> >> __________________________________________ >> Maria Håkansson, Ph.D. >> Senior Scientist, Max-lab, Lund University >> Phone: +46 (0) 76 8585 706 >> Fax: +46 (0) 46 222 47 10 >> Ole Römers väg 1 (P.O. Box 188) >> SE-221 00 Lund, Sweden >> >> Web address: www.maxlab.lu.se <http://www.maxlab.lu.se> >> Email: [log in to unmask] <mailto:[log in to unmask]> >> __________________________________________ > > > > -- -- Dr. Jeroen R. Mesters Gruppenleiter Strukturelle Neurobiologie und Kristallogenese Institut für Biochemie Universität zu Lübeck Zentrum für Medizinische Struktur- und Zellbiologie Ratzeburger Allee 160, D-23538 Lübeck Tel: +49-451-5004065 Fax: +49-451-5004068 Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.selfish-brain.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --