Bead Mill

Contents

1. Freezing Yeast
2. Operating the Bead Mill
3. Reserving the Bead Mill

1. Freezing Yeast

1. Grow Yeast as per your protocol.
2. Harvest yeast in 1L bottles.
3. Resuspend Yeast in water and transfer to 50 mL conical tubes.
4. You can either pellet and freeze at -80C here or move to freezing in “balls.”
5. To freeze in balls, resuspend the pelleted yeast in a very small amount of your Lysis Buffer (contains buffer, salt, etc. as well as PIs (HALT or cOmplete) and PMSF). I recommend starting at ~2 mL per Liter of yeast and mix/vortex to make the yeast very slightly fluid. Add more Buffer if pellet is still solid.
6. Transfer the lightly suspended pellet to a 30 mL syringe (with the end of the syringe blocked with parafilm). You want the pellet to be viscous enough to not flow through the hole without pressure.
7. Get a 250 mL plastic beaker or greater and place in a styrofoam container filled with liquid Nitrogen (also fill the plastic container ~half full with liquid Nitrogen).
8. Remove the parafilm and force the pellet though into the beaker containing liquid Nitrogen. Can make little frozen yeast balls that can be easily transferred to a 50 mL conical.
9. Yeast balls are then stored at -80C.

2. Operating the Bead Mill

Here is a link to the manual. Specifically look to page 29 to familiarize yourself with how to operate the locking mechanism.

Make sure to have a full dewar of liquid Nitrogen. This protocol takes a lot.

I do five cycles of three minutes on at 15 Hz (1/s). Freeze the cylinders in liquid Nitrogen inbetween cycles.

There may be an upper limit of how much pellet can be loaded in a single chamber per run, but I am not sure what it is. Currently I have loaded ~7.5g of pellet in one chamber and had successful breaking.

I pick the yeast powder out of the chamber using a 200 uL tip and then knocking the powder in a 50 mL conical suspended in Liquid Nitrogen (no thawing allowed, if there is some, we have PIs in the pellet as well).

The lysed yeast can be stored in the freezer indefinitely.

To check that the yeast is lysed, take a small bit of yeast powder and place in 20 uL of water. Pipet 5 uL of suspension on a glass slide and cover with a coverslip. Examine the cells under a light microscope (I prefer the one of Steve’s desk). See image for the difference between intact and lysed yeast. Lysed yeast are distorted and appear darker.

The process is loud, so plan maybe plan the lysis during off hours to keep everyone happy.

3. Reserving the Bead Mill

The Bead Mill is a Core piece of equipment currently house in Dr. Arlen Johnson’s lab (NMS 1.126).

Currently the equipment is reserved by going to the lab and checking it out on a paper calendar.

If you want to ensure that it is available, then reserve ahead of time. If you want it over the weekend, check it out Friday.

Jeff (2016_10_11)