McClean: Glycerol stocks (yeast)

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Revision as of 14:32, 12 October 2012 by Megan N McClean (Talk | contribs)
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Contents

Overview

Yeast strains can be maintained almost indefinitely at -80°C in glycerol stocks. Here’s how:

Materials

  • 30% Sterile glycerol solution
  • Cryotubes (Fisher #12-565-163N; Nunc #375418)
  • Cryotube Caps, Yellow (Fisher #12-565-246; Nunc #355077)

Instructions

Note: You are going to freeze down TWO vials of culture, one for the main -80°C stocks and the other for the backup -80°C stocks.

Day 1

  1. Add the strain to the McClean lab database and assign it a yMM ID. If this is your first time entering something into the database, please talk to Megan. Some members of the lab will not have access to the main database, in which case you should ask Megan to create the database entry for you.
  2. From a plate, pick a SINGLE COLONY.
  3. Inoculate the cells overnight in YPD (or selective media if they contain plasmids) and grow at 30°C so that in the morning you have a saturated culture.

Day 2

  1. Put 900 μl of the sterile 30% glycerol into two cryovials (900μl per each vial).
  2. Add 900 μl of the saturated overnight culture to each cryovial.
  3. Invert the vials several times to mix.
  4. Put a yellow cap on each vial. Label the top of each cryovial with just the yMM number (i.e. '100') and label the side of each tube with the yMM number, the date, your initials and any other useful information.
  5. Add one tube to the appropriate yMM stock box in the main -80°C and add the other vial to the backup stock box. Do NOT use the -20°C freezer, glycerol stocks do not maintain viability above about -55°C.
  6. Double check that the vials are labeled well and entered into the McClean lab database!

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
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  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Current Protocols in Molecular Biology. V1 Section 1.8.2

Contact

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