McClean: Glycerol stocks (yeast)
Yeast strains can be maintained almost indefinitely at -80°C in glycerol stocks. You need to make sure that as soon as you have a confirmed strain (confirmation by colony PCR, sequencing, mating type checking, etc) you freeze it down as a glycerol stock in the main lab database.
- 30% Sterile glycerol solution
- Cryotubes (Fisher #12-565-163N; Nunc #375418)
- Cryotube Caps, Yellow (Fisher #12-565-246; Nunc #355077)
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.
- 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.
- From a plate, pick a SINGLE COLONY.
- 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.
- Put 900 μl of the sterile 30% glycerol into two cryovials (900μl per each vial).
- Add 900 μl of the saturated overnight culture to each cryovial.
- Invert the vials several times to mix.
- 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.
- 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.
- Double check that the vials are labeled well and entered into the McClean lab database!
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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- Megan N McClean 14:36, 12 October 2012 (EDT) Failure to keep glycerol stocks and poor database maintenance are the two biggest lab no-nos. There should never be a blank spot in the -80°C stock boxes. You should never be performing multiple experiments with a strain that isn't glycerol stocked and entered into the database. Enter as much information as possible about your strain when you put it in the database (including references to notebook pages, etc) so that in the future lab members can understand how the strain was created and use your strain with confidence.
Current Protocols in Molecular Biology. V1 Section 1.8.2
- Megan N McClean 14:01, 12 October 2012 (EDT)
or instead, discuss this protocol.