DLadakis, The seed stock solution should be your crystallization solution with a slight (5% maybe) increase in the precipitant. In my experience, I have not needed additional protein to stabilize the nano-crystals, but as with all things, every protein is different.
The idea is the crystals should be stable in the stock solution and not dissolve. I generally place one crystal in 500 microL of seed stock (with the little ball for crushing). Once I've vortexed for 5 minutes and a 13,000 rpm centrifuge spin for 5 minutes, I then prepare 3 1000-fold dilutions (i.e. 1000-fold, 1 million fold and 1 billion fold). All can be stored at -20 degrees C for extended periods of time. When I setup the drops I generally either add 0.2 microL to a protein + well drop (assuming 2 microL drop), or instead of adding the well solution, I add the seed stock (or dilution) in equivalent volumes to the protein. Another option is streak seeding, where you streak a cat (horse, etc) whisker (or purchase the Hampton Research equivalent) across a crystal, then streak the whisker through your crystallization drops, with each successive drop being a dilution of the first streak (4 - 6 drops per streak). Hope this helps, Kelly Daughtry <http://walk.avonfoundation.org/site/TR?px=7043512&pg=personal&fr_id=2260&s_src=BF_emailbadge> ******************************************************* Kelly Daughtry, Ph.D. IRTA Fellow Mechanisms of Mutation Group Molecular Genetics Laboratory, MD E-301 National Institute of Environmental Health Sciences 111 TW Alexander Drive Research Triangle Park, NC 27709 Tel. (919) 541-3452 ******************************************************* On Thu, May 30, 2013 at 5:15 AM, dladakis <[email protected]> wrote: > Dear all > I would like your advice on preparing a seed stock for microseeding. > Should the seed stabilizing stock soltion include soluble protein as well. > If it doesn't will the seeds eventually disolve leading to an unsuccesfull > screen? > Also how is the seedstock supposed to look under the microscope, clear? > Thanks for your help > DLadakis >
