OT cryogenic stocks (based on Nigel Grimsley’s protocol) Revised 20131220


FREEZING


Equipment & reagents

  • Cryovials, 2ml, screw-cap

  • Cryobox (or cryocane)

  • Cryotongs (tongs w/ rubber tips or wrapped w/ tubing)

  • Sterile DMSO or sterile-filtered DMSO (Use 0.22µm nylon filter)

  • OT cells at late log phase (OD600 ~ 0.2 - 0.4, should be green, not pale)

  • ASW (artificial seawater), pre-warmed to room temp

  • (Optional) 1,000x KNP antibiotics mix

  • LN2 in 4L bottle

  • Wide-bottomed bowl for LN2

  • Long-term storage dewar for frozen cells


Preparation

  1. Label all cryovials with LN2-resistant lab marker.

  2. Submerge bottom half of cryobox in LN2 bowl and cool in LN2.

  3. Prepare fresh 22% DMSO (2.2ml DMSO + 7.8ml ASW). Enough for ~ 10 vials.

  4. Aliquot 1ml 22% DMSO/ASW into each cryovial.


Freeze

  1. Add 1ml of late log-phase OT cells.

  2. Mix by gentle up+down pipetting or inversion.

    • Don’t let cells sit on the bench in 11% DMSO too long.

  3. Pick up cryovial with cryotongs and snap-freeze in LN2.

    • Do NOT submerge cryovial in LN2 or it might explode due to rapid expansion of seawater. Instead, hold the cryovial cap with the cryotongs and immerse the bottom portion in LN2 and hold until the boiling stops. Cooling is slower, but allows the cryovial cap to adapt to the vial’s shape change.

  4. Load cells into cryobox.

  5. Store in LN2 tank and record rack# and row# in the cell lines inventory.


Test the quality of freezing - do for every freezing experiment

Wait 1 week, then thaw 1 vial of cells (see protocol below) to make sure they’re revivable.


THAWING


If you thaw the second-to-last tube of a strain, please freeze away some more cells when the newly ‘restored’ culture is nice and healthy. Otherwise, we’ll risk to rely on the absolute last tube.


Equipment & Reagents

  • LN2

  • Small dewar for LN2

  • H2O bath @ 37 - 40°C (for thawing cells)

  • ASW, 50ml in culture flask, pre-warmed to room temp.


Procedure

  1. Remove one (or more) vial of cells from the LN2 storage tank; keep submerged in LN2 up until thawing.

  2. Thaw one vial of cells in 40°C water bath.

  3. When most of the ice has melted, dilute the entire vial’s contents into 50ml ASW.

    • Don’t thaw completely; it’s better if there are some frozen bits floating around

  4. In ~ 1 week, cells should appear green by eye.