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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:
[log in to unmask]" type="cite">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  
__________________________________________






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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
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If you can look into the seeds of time and say which grain will grow and which will not - speak then to me  (Macbeth)
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