"
Team:Evry/Notebook/Protocols/CompetentsM9
From 2014.igem.org
(Difference between revisions)
| (2 intermediate revisions not shown) | |||
| Line 3: | Line 3: | ||
<div align="center"> | <div align="center"> | ||
<FONT COLOR="blue"> | <FONT COLOR="blue"> | ||
| - | < | + | <h4>Electro-competent <i>Pseudovibrio</i> in M9-CASA +3%NaCl</h4> |
</FONT> | </FONT> | ||
</div> | </div> | ||
<br><br> | <br><br> | ||
| + | |||
<div align="left"> | <div align="left"> | ||
| - | < | + | <div align="justify"> |
| + | |||
| + | <i><b>Reagents and Materials</i></b><br> | ||
| + | <br> | ||
| + | |||
| + | * Refrigerated centrifuge (4°C)<br> | ||
| + | * M9-CASA+3%NaCl 1X<br> | ||
| + | *Cold glycerol 10% (4°C)<br> | ||
| + | *Cold sorbitol 2% (4°C)<br> | ||
| + | <br> | ||
| + | |||
| + | <i><b>Experimental procedure</i></b><br> | ||
| + | <br> | ||
| + | |||
| + | -Make a pre-culture of Pseudovibrio in 3mL of M9-CASA+3%NaCl 1X and let it incubate overnight at 30°C with shaking.<br> | ||
| + | <br> | ||
| + | |||
| + | -Relaunch the Pseudovibrio culture in 100mL of M9-CASA+3%NaCl 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 or cold sorbitol three 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 1)<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> | ||
| + | |||
| + | |||
| + | |||
</div> | </div> | ||
| + | </div> | ||
| + | |||
| + | |||
| + | |||
</htlm> | </htlm> | ||
Latest revision as of 00:45, 18 October 2014
Electro-competent Pseudovibrio in M9-CASA +3%NaCl
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.
* 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.
