— Olivia Heath 2018/05/03 17:28

When we obtain or create a new yeast strain, the strain should be IMMEDIATELY frozen down and added to the collection of strains in the -80°C Freezer. AT THE SAME TIME, the complete strain information must be added to the lab yeast strains spreadsheet, MM-Yeast-Strains.ods, which is in the Dropbox “Yeast Strains” folder.

80% Glycerol

3/8" ToughSpot Stickers

Screw Top Tubes

Niblets

0. In order to freeze down yeast strains, you must first grow them up overnight in liquid media, including selecting for any appropriate markers. See Overnight Yeast Strains for the protocol. This step must be started 18-24h before the following steps.

1. Enter information about the strain in the yeast strains spreadsheet.

• Open the “MM-Yeast-Strains.ods” spreadsheet located in the “Yeast Strains” folder of the Dropbox.
• In the spreadsheet enter all required information for every column, for the new strain number e.g. “MM####”.
• Any information at all about the strain that does not go on one of the other columns should be included in the “Notes” column.

2. Appropriately label the tube that the new strain will go in.

• Get a screw top tube, a niblet, and three 3_8_toughspot_stickers per strain that you are freezing down.
• There should be pre-printed stickers with consecutive MM strain numbers starting at the next available number. If not, you can print more or hand-write three using a fine point black sharpie.
• Apply one sticker to the side of the tube, and one sticker directly to the top of the tube. Then snap the clear niblet in place in the top of the tube cap, and apply the third sticker on top of the niblet, covering the first sticker.

3. Freeze the strain

• Using a P 1000 and a new 1000, ut 330 μl of 80% glycerol into each screw top tube, one per strain you are freezing.

• Note: 80% glycerol is very viscous, please pipette slowly. μl

• Using a 1000μl pipet and another new 1000μl Barrier tip, add 990 μl of yeast suspended in liquid yeast media, directly from your overnight culture from step 0, into the appropriately labeled tube already containing the 80% Glycerol.

5. Close the screw top tube securely and vortex the screw top tubes to ensure everything is mixed. Then put the tube in the appropriate place in its -80C cardboard storage box in the racks in the -80°C Freezer and put in -80°C Freezer in the appropriately labeled box.

• Please see the -80 freezer map outside of -80°C Freezer to determine which box the strain will live in according to its strain number.
• If a box is full, label all four sides and the top of a new box using laser printed labels covered in Packing Tape. Your labels should match the older box labels exactly except for the strain numbers. There is a box marked 'Yeast Box Labels' that contains pre-made labels for the boxes.

• Note: it is easier to place label on box if you first stick label on the bench, then cut a piece of tape and then stick center of tape to center of label and afterward place label on box.

NOTE: When finished with strains, confirm that they are not being used for anything other than freezing down today. If not, it is not necessary to place them back in the shaker. Unless otherwise indicated, they are ready for disposal. If you have not learned how to properly dispose of yeast, ask someone in the lab to teach you one-on-one before proceeding.