Team:Hawaii/Protocols/Cryopreserve cells

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Contents

Purpose

Cryopreserve bacteria for long term storage in -80C deep freezer.

Materials

  • Cryovials
  • Cryoboxes
  • Mr. Frosty (optional)

Reagents

  • General Bacteria
    • 80% Glycerol Stock
    • Growth Medium
  • Cyanobacteria
    • DMSO solution
    • 1x BG-11
    • H20

Equipment

  • -80C Freezer

Procedure

Freezing

  1. Label (strain, date, medium) on cryovials, do at least 3 vials /strain.
  2. E. coli (26% glycerol is higher than normal E. coli cryo-preservation protocols, this concentration allows easy scraping of top without thawing out entire tube according to Dr. Callahan)
    1. dispense 0.4mL 80% sterile glycerol stock into cryovial
    2. add 0.8mL E. coli culture grown overnight in TB medium, so final glycerol concentration in vial is ~26%
    3. perform quick freeze (prevent water-molecules to organize and form large crystals and rupture membrane) by immediately putting cryotube into eppendorf rack holes prefilled and -80C prechilled w/ 70% ethanol. Place rack in -80C for freezing.
    4. after freezing is complete in -80C, transfer vials to final cryoboxes at -80C
    5. Record cryopreserved bacteria addition in -80C Deep Freezer Inventory.

Thawing

  1. Record cryopreserved bacteria removal in -80C Deep Freezer Inventory.
  2. Use one of the following thawing methods:
    1. General Bacteria: Waterbath Method
      1. Thaw quickly in 37C water bath.
      2. Cells are immediately pelleted by centrifugation of the cryovial, and the supernatant is discarded. One ml of fresh growth medium is placed into the vial to resuspend the pellet.
      3. Dispense contents on media agar plate or in liquid media.


Notes

According to a [http://www-cyanosite.bio.purdue.edu/protocols/cryo.html writeup] (local mirror) by Jerry Brand at UTEX

[3] Although glycerol is an effective cryoprotective agent for many bacteria, it is not effective for most cyanobacteria. Methanol at approximately 5% (v/v) is suitable for most strains. However, we have been successful with concentrations of methanol ranging from 2% to 12.5%, and DMSO ranging from 4 to 15 %, depending on the culture. A small fraction of some cultures survive with no added cryoprotective agent.
[5] Cells are killed by exposure to bright light when in cryoprotective solution. Keep the culture in subdued room light while handling, and in complete darkness at other times.

Acknowledgments

References

See [http://openwetware.org/wiki/IGEM:Harvard/2006/Cyanobacteria/Protocols Harvard iGEM 2006 Cyanobacteria Protocol]

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