"

From 2014.igem.org

Reagents and Materials

* Refrigerated centrifuge (4°C)
* M9-CASA+3%NaCl 1X
*Cold glycerol 10% (4°C)
*Cold sorbitol 2% (4°C)

Experimental procedure

-Make a pre-culture of Pseudovibrio in 3mL of M9-CASA+3%NaCl 1X and let it incubate overnight at 30°C with shaking.

-Relaunch the Pseudovibrio culture in 100mL of M9-CASA+3%NaCl 1X.
-Let it grow in a 30°C shaking incubator until DO (600nm) = 1.2
-Divide the culture into two 50mL Falcon tubes.
-Wash cells with cold glycerol or cold sorbitol three times
(Centrifuge at 4000g, 4°C for 10min and re-suspend):
* 50 mL (x 1)
* 25 mL (x 1)
* 5 mL (x 1)
-Re-suspend a last time washed cells in 500µL of glycerol 10%.
-Aliquot 50µL of competent cells and stock them at -80°C for maximum three weeks.