McClean:E. coli Glycerol Stocks

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


  • 65% Bacterial glycerol solution (see McClean: Bacterial Glycerol 65%)
  • Cryotubes (Nunc Cat #12-565-163N or Fisher Cat #375418)
  • Cryotube Caps, Blue (Nunc Cat #12-565-243 or Fisher Cat #354879)


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 bMM ID. Also enter information for the plasmid contained in the bacterial strain (if relevant) and make sure that the two are appropriately linked in the database. If this is your first time entering something into the database, please talk to Megan.
  2. From a plate, pick E. coli from a SINGLE COLONY.
  3. Inoculate the cells overnight in LB plus the appropriate selective drug (for plasmid maintenance) so that in the morning you have a saturated culture. Grow on the roller drum in the 37°C warm room

Day 2

  1. Put 900 μl of the sterile bacterial 65% glycerol into two cryovials (900μl per each vial).
  2. Add 900 μl of the saturated overnight bacterial culture to each cryovial.
  3. Invert the vials several times to mix.
  4. Put a blue cap on each vial. Label the top of each cryovial with just the bMM number (i.e. '100') and label the side of each tube with the bMM number, the date, and your initials.
  5. Add one tube to the appropriate bacterial 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!


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

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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Current Protocols in Molecular Biology. V1 Section 1.8.2


or instead, discuss this protocol.