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Artem Evdokimov: 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.

 artem.evdokimov@GMAIL.COM

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!

ARCHIVE: Introduction, Pre-crystallization, Crystallization, Post-crystallization, Derivatization, Cryoprotection, Diffraction, Symmetry, Structure Solution, Structure Refinement, Structure Analysis & Presentation.