Freezing -80 Stocks

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E. coli Stocks

Day 1

  • Streak cells from a cloning experiment onto a plate with antibiotics, in the case of a strain that should have a plasmid that confers antibiotic resistance.
  • Incubate plate overnight at 37°.

Day 2

  • Do the resulting colonies look correct? Is there contamination anywhere on the plate? If so, re-streak.
  • Sterilely put 10ml of LB with appropriate antibiotics in a sterile Erlenmeyer tube.
  • Add one colony of the strain.
  • Incubate in the shaking incubator overnight at 37°.


Day 3

  • Create a label for the stock using the label maker in the write-up area
  • Update the online strain database
  • Label a freezer tube with the printed side and top label.
  • Fill the freezer tube with 20% final concentration in media. This is then a 1:3 (400ul 80% glycerol; 1200ul culture) for final glycerol concentration of 20%.
  • Vortex and immediately put in the -80 freezer in the appropriate box / location.


Streptococcus sp. Stocks

Day 1

  • Streak cells from the original stock (either from a previous -80° stock or from a cloning experiment). Use a blood plate for this.
  • Incubate plate overnight at 37°.

Day 2

  • Do the resulting colonies look correct? Are they all one colony type? Did they lyse blood, where appropriate?
  • Pick one representative colony and 'cross streak' the sample with a sterile cotton swab to create a lawn on a blood agar plate. A half plate can be used, in the case of freezing multiple strains.
  • Incubate plate overnight at 37°.

Day 3

  • Create a label for the stock using the label maker in the write-up area
  • Update the online strain database
  • Label a freezer tube with the printed side and top label.
  • Fill the freezer tube with 20% glycerol in Trypic Soy Broth (S. pneumoniae and others) or Todd-Hewitt Broth (S. suis). This is then a 1:3 (300ul 80% glycerol; 900ul Trypic Soy Broth) for final glycerol concentration of 20%. Notice that this is a different volume than E. coli liquid cultures.
  • In the biological hood, use a sterile cotton swab to collect as much of the growth on the plate as possible. Avoid any chance of getting in other strains, specifically when using half-plates with two strains. It is better to leave colonies than risk any strains being mixed.
  • Swish the swab in the tube extensively; pull the swab out 3/4 way, and wring it out on the side of the tube.
  • Vortex tube and immediately put in the -80 freezer in the appropriate box / location.