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Making Seed Stock

Preparing seeds for use in C3

  1. Identify a suitable condition from which to prepare seeds – this should be a drop that contains crystals and not too much precipitate.  If both subwells in a multi-subwell plate have crystals, use can use both subwells as your seed source.  If you have only a small number of crystals in any one drop, but have a number of crystal-containing drops all under similar conditions, then combine the droplets to make the seeds.  You need to have a reasonable amount of crystal mass from which to make seeds – a single flyspeck-sized crystal in a low volume sitting drop is not enough
  2. Make up 1ml of the reservoir solution associated with the seed source drop (‘reservoir’). If you are combining different drops, then make up a reservoir solution that is at least as concentrated as the most concentrated seed source drop reservoir.
  3. Put a seed-bead (1/8” Teflon bead, or a few (ten?) smaller ceramic, glass or stainless steel beads) into a 1.5ml Eppendorf tube. Label the seed-bead Eppendorf tube (barcode, well, date), and add 50μl of the reservoir solution to it.
  4. Cut the tape from the seed source drop – add 1μl of the reservoir solution to the droplet, and then poke a crystal with one of the Hampton tools. The crystal should crush when you poke it. If it doesn’t, then the crystals are too old to make good seeds, and you should select another candidate for your seed source. If the crystals start dissolving when you add the 1μl of reservoir, you will need to make up a new seed stock reservoir with more precipitant in it, and try again.
  5. Using a 2μl pipette, move the seed source droplet, and all the crystals, to the 50ul of reservoir solution in the seed bead Eppendorf tub.
  6. Close the lid of the tube, and vortex at full speed for a minute.
  7. Clean the Hampton tool (rinse it with water, then wipe it with ethanol), and replace it in the toolbox.
  8. Add another 200μl of the reservoir solution to the vortexed Eppendorf, and vortex briefly (2 seconds) to mix. This is your seed stock.
  9. Using the Mosquito, set up a plate with a seeding protocol. The standard protocol will have 150nl protein, 100nl reservoir, and 50nl of your seed stock. The seed stock may have to be further diluted, or the drop ratio changed to get good crystals from the seed stock.

The seed-beads can be obtained from B J Bearings (13 Endevour St. Warragul, VIC 3820, telephone 03 5623 5444) – 1/8” Delrin Gr1 Balls – product code KU.3.175DELRIN – about $20 for 100.

Reference: J.R. Luft and G.T. DeTitta. (1999). A Method to Produce Microseed Stock for Use in the Crystallization of Biological Macromolecules, Acta Cryst., D55, 988-993