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Team:Evry/Notebook/Protocols/CompetentsMB
From 2014.igem.org
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| + | <i><b>Reagents and Materials</i></b><br> | ||
| + | <br> | ||
| + | |||
| + | * Refrigerated centrifuge (4°C)<br> | ||
| + | * Marine Broth 1X<br> | ||
| + | *Cold glycerol 10% (4°C)<br> | ||
| + | <br> | ||
| + | |||
| + | <i><b>Experimental procedure</i></b><br> | ||
| + | <br> | ||
| + | |||
| + | -Make a pre-culture of Pseudovibrio in 3mL of MB 1X and let it incubate overnight at 30°C with shaking.<br> | ||
| + | <br> | ||
| + | |||
| + | -Relaunch the Pseudovibrio culture in 100mL of MB 1X.<br> | ||
| + | -Let it grow in a 30°C shaking incubator until DO (600nm) = 1.2<br> | ||
| + | -Divide the culture into two 50mL Falcon tubes.<br> | ||
| + | -Wash cells with cold glycerol 10% five times <br> | ||
| + | (Centrifuge at 4000g, 4°C for 10min and re-suspend):<br> | ||
| + | |||
| + | * 50 mL (x 1)<br> | ||
| + | * 25 mL (x 1)<br> | ||
| + | * 5 mL (x 3)<br> | ||
| + | -Re-suspend a last time washed cells in 500µL of glycerol 10%.<br> | ||
| + | -Aliquot 50µL of competent cells and stock them at -80°C for maximum three weeks.<br> | ||
| + | <br> | ||
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Latest revision as of 00:45, 18 October 2014
Electro-competent Pseudovibrio in Marine broth
Reagents and Materials
* Refrigerated centrifuge (4°C)
* Marine Broth 1X
*Cold glycerol 10% (4°C)
Experimental procedure
-Make a pre-culture of Pseudovibrio in 3mL of MB 1X and let it incubate overnight at 30°C with shaking.
-Relaunch the Pseudovibrio culture in 100mL of MB 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 10% five times
(Centrifuge at 4000g, 4°C for 10min and re-suspend):
* 50 mL (x 1)
* 25 mL (x 1)
* 5 mL (x 3)
-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.
* Refrigerated centrifuge (4°C)
* Marine Broth 1X
*Cold glycerol 10% (4°C)
Experimental procedure
-Make a pre-culture of Pseudovibrio in 3mL of MB 1X and let it incubate overnight at 30°C with shaking.
-Relaunch the Pseudovibrio culture in 100mL of MB 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 10% five times
(Centrifuge at 4000g, 4°C for 10min and re-suspend):
* 50 mL (x 1)
* 25 mL (x 1)
* 5 mL (x 3)
-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.
