Small crystals or microcrystals can be harvested from the Crystal Former to make a seed stock.
Rescuing seeds from the Crystal Former
Preparing to harvest the seed stock
- Prepare your stabilizing solution, which is typically your crystallization condition with slightly higher precipitant concentration.
- Chill your stabilizing solution and vial on ice.
- Place a small drop of water on a glass coverslip or a lid of the plate.
- Cut the tabs connecting the individual channel seals of the Crystal Former backing (see harvesting video).
- Make sure the cuts are complete to avoid disturbance of the neighboring channels.
- Use fine forceps to lift up the backing and flip it over, so the surface in contact with the channel is facing up.
- Place the backing film onto a drop of water to “glue” it to the coverslip of plate lid, so that it does not move around during harvesting.
- Immediately apply 10-20 µL of the chilled stabilizing solution to the backing (tracing the U-shape of the channel), then to the open channel.
- Harvest the crystals from the backing first. Move the tip of the pipette along the U-shape on the backing to dislodge any crystals that may be stuck to the film.
- Aspirate the solution back into the tip and place it in the chilled collection vial.
- Repeat the process with the channel.
- You may need to use crystal manipulation tools to dislodge any crystals suck to the walls of the channel.
- Aspirate the solution with loose crystals from the channel by placing the pipette tip into the precipitant inlet.
Seed stock preparation
- Crush the crystals using the glass rod with a tip melted into a small bulb, or use glass beads and a vortex.
- You can make your seed stock dilutions, aliquot them, and freeze for later use.
Seeding into the Crystal Former
Crystal Formers can be used for conducting seeding experiments. Simply mix your seed stock with your protein solution and proceed to set up the plate as usual. Optional step involves placing the microliter plate with screen solution aliquots in refrigerator to chill the screen solutions prior to plate setup. Manual setups will allow to keep the plate on ice, while robotic setups should be quick enough to preserve the seeds. Seeding can be done into the grid screen around the initial hit condition, or into the second sparse matrix screening pass, potentially identifying new crystallization conditions.