Team:Hawaii/Protocols/Cryopreserve cells

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

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)
 * 3) dispense 0.4mL 80% sterile glycerol stock into cryovial
 * 4) add 0.8mL E. coli culture grown overnight in TB medium, so final glycerol concentration in vial is ~26%
 * 5) 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.
 * 6) after freezing is complete in -80C, transfer vials to final cryoboxes at -80C
 * 7) 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:
 * 3) General Bacteria: Waterbath Method
 * 4) Thaw quickly in 37C water bath.
 * 5) 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.
 * 6) Dispense contents on media agar plate or in liquid media.