"
Team:Hawaii/PCC6803 Cryostock Thaw Test
From 2008.igem.org
Contents |
Experiment Title
- Thaw PCC6803 -80C deep frozen stock to ensure that our cryogenic protocol works. This will ensure that we will have re animatable Synechocystis cells from deep freeze in-case everything dies.
Methods
- 2008.05.09 Cryogenically frozen PCC6803 cells using the methods prescribed [http://www-cyanosite.bio.purdue.edu/protocols/cryo.html here] from UTEX.
Cultures to be revived are removed from liquid nitrogen storage and warmed rapidly to room temperature[8]. Cells are immediately pelleted by centrifugation of the cryovial[9,10], and the supernatant is discarded. One ml of fresh growth medium is placed into the vial to suspend the pellet. The cryovial lid is slightly loosened to allow gas exchange, and the contents of the vial are kept in complete darkness for 1-2 days. The culture can then be placed on agar or in liquid growth media under normal growth conditions. The viable cells should begin normal growth within 1 - 2 days in light, although they are especially susceptible to damage by excessive light intensity for the first day or two of illumination.
- 2008.06.04 Thawed frozen stock from 5/9, spun down, added fresh BG-11 media (+thiosulfate, +TES pH 8.0 adjusted). Incubate in cryotube in complete darkness (aluminum foil wrapped) @ 26C for two days.
- 2008.06.06
- Method #1: Plated cryo-tube content using streak and hockey puck methods
- Method #2: Liquid culture by adding 500 uL cryo-tube content to 10 mL BG-11 in T75 flat rectangular tissue culture flask. Shaken at 100rpm using Innova 2050.
Results
7days from initial thaw on 6/4
- Liquid Stock (low cell density)
- slightly grew back on plate
- almost no growth in liquid
- Scraped Stock (high cell density)
- grew back on plate
- growth to light green in liquid
Discussion
The thawed PCC6803 grew back on plates for both liquid and scraped stock. Little growth was observed in liquid for the the liquid stock (low cell density), most likely that not high enough density cells were inoculated in the 10mL media. 5% of total volume (e.g. 0.5 mL in 10 mL fresh media) is about the lowest amount to inoculate to grow back in a reasonable amount of time (2wk) (in no CO2, no Glucose, +TES pH8, +thiosulfate, 24hr light, 26C)
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
