Serial Dilution

1. Fill sitting drop well with 50 µl stabilizing solution
2. Place 1 crystal in sitting drop well
3. Crush crystal with 25 gauge needle or micro-tools
4. Pipette entire volume into 1.5 ml eppendorf tube
5. Spin 30 sec on high to remove macroscopic crystals
6. Serially dilute and vortex micro-crystals in stabilizing solution to have dilutions of 1, 10, 100, and 1000
7. Add to four pre-equilibrated drops 1/4-1/3 the drop’s volume of one of the serially diluted micro-crystal solutions.
8. Re-seal coverslips over original wells

Fiber transfer

1. Invert coverslip with native crystal (Optional: crush crystal)
2. Pass tip of cat whisker (or similar small fiber) through drop, touching crystals
3. Streak tip of whisker successively through 3 or 4 pre-equilibrated drops
4. Re-seal coverslips over original wells

Successive streaking with a fiber is effectively a serial dilution.
Pre-equilibrated drops contain protein and precipitant that is at a concentration just below what is required for precipitation or crystallization of the protein. These drops should incubate for at least 2 hours, hanging from coverslips that are sealed over a well containing precipitant (normal hanging drop experiment).