By popular request here's my favorite version of the in-screen seeding. We use a Mosquito but it doesn't have to be a specific robot as long as it can dispense relatively tiny volumes of seed stock.
Caveats: (1) if I am desperate enough to do this, then the situation is pretty bad indeed and I don't mind wasting some protein (2) my success rate is not hugely favorable but this does work on occasion when other things have failed (1) identify a few likely conditions. Ideally they have microcrystals but desperation has made me try 'lovable precipitates' in the past, with a modest degree of success. (2) harvest entire drop using a few ul of mother liquor as diluent (3) break the existing crystals using your favorite method (sead beed, etc.) mine involves swirling a pipette tip in the mixture, running it along the walls, with rapid pipetting up and down. Dilute seed stock to useful volume (enough for screening). (4) I do not normally centrifuge the resulting seed stock, but some people do (5) dispense your screen as always with the usual protein/reservoir ratio. Let's say you like drops of the 0.2ul+0.2ul variety - add 25 nanoliters of the seed stock *last*. Optional mixing of the condition is a fun thing to try but it seems not to matter very much. Note that I typically use the same tips to dispense seed stock, fully aware that this causes cross-contamination of conditions. I don't mind :) (5a) variation - add seed stock to protein, then dispense ASAP. Surprisingly not a bad option, practically speaking. (5b) variation - crosslink seed stock very gently in solution (with trace of glutaraldehyde) before use. Buffers/additives with primary or secondary amine groups do interfere, of course. (5c) variation - mix seeds from SEVERAL initial hit conditions, then use as one seed stock. Be ready for fireworks as they may not be compatible! (6) endure nail-biting wait for results :) As noted earlier, it's not a sure-fire way to get new hit conditions but it does seem to work and it's a fun way to put to use a remainder of otherwise useless protein (when you've tried all other tricks you like to try). Comments and suggestions are always welcome! Artem On Fri, Oct 28, 2011 at 1:55 PM, Artem Evdokimov <[email protected]>wrote: > I would be glad to share ours. > > Artem > On Oct 28, 2011 1:29 PM, "Watson, Randy" <[email protected]> wrote: > >> Hi all, >> >> I am trying to optimize crystals and have heard of a technique where one >> can prepare a seedstock from existing crystals and use it to broadly >> re-screen from scratch for hits in new conditions. >> >> I have access to a mosquito robot and was wondering if anyone has a >> protocol or recommendations on how to go about doing this using a robot for >> screening. >> >> Thank you! >> Randy Watson >> >
