Team:Evry/Protocols/01

From 2013.igem.org

(Difference between revisions)
Line 2: Line 2:
<html>
<html>
-
<a href='https://2013.igem.org/Team:Evry/Protocoles/01' title='Vers la page française'> <img src='https://static.igem.org/mediawiki/2013/b/b9/Francais.jpg'/></a>
+
<a href='https://2013.igem.org/Team:Evry/Protocoles/03' title='Vers la page française'> <img src='https://static.igem.org/mediawiki/2013/b/b9/Francais.jpg'/></a>
-
<h1 align='center'> Competents cells </h1>
+
<h1 align='center'> Plasmid purification </h1>
-
First prepare the following solutions required to make competent cells:<br/>
+
<i>Protocole from Macherey-Nagel plasmid purification notebook</i><br><br>
-
Solution for 1M Cacl2:<br/>
+
-
Add 14,30g of CaCl2 into 100 ml desalted water<br/>
+
-
<br/>
+
-
Solution for 0,1M Cacl2:<br/>
+
<b>. Récupération des bactéries</b><br>
-
Add 50 mL of CaCl2 1M solution into 450 ml of desalted water<br/>
+
Set saturated E.coli LM culture into 2 mL tubes. Centrifuge at 11 000 x g for 30 secondes. Discard as much as supernatant as possible.<br><br>
-
<br/>
+
-
Solution for 0,1M Cacl2 + 15% glycerol:<br/>
+
<b>.Cell lysis</b> <br>
-
Add 50 mL of CaCl2 1M solution and 75 mL of glycerol 100% into 450 ml of desalted water<br/>
+
Add 250 μL Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette.
-
</p>
+
<br>
-
<br/>
+
Add 250 μL Buffer A2 (lysis buffer). Mix gently by inverting the tube 6 - 8 times. Incubate at room temperature until lysate appears clear.<br>
-
<p>For 200 ml LB medium, add 400 µL of strain sample.<br/>
+
Add 300 μL Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .<br>
-
Let the bacteria grow until it reaches an OD between 0,3 and 0,35.<br/>
+
 
-
Once it reached the right OD, put the medium on ice for 30 minutes to slow down growth.<br/>
+
<b>. Clarification of lysate</b><br>
-
Split the 200 mL into 4x50 mL tubes then centrifuge at 3000 rpm for 5 minutes at 4°C and suppress supernatant afterwards.<br/>
+
Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.<br><br>
-
Resuspend the cells with 5 mL of Cacl2 at 0,1M for each 50 mL tube.<br/>
+
 
-
Again, put the medium on ice for 30 minutes.<br/>
+
<b>.Bind ADN<br></b>
-
Centrifuge at 3000 rpm for 5 minutes at 4°C then suppress supernatant.<br/>
+
Place a NucleoSpin Plasmid Column in a Collection Tube of 2 mL et and set the supernatant from the last step. Centrifuge at 11 000 x g for 1 minute. Discard flow-through and place the column back into the collection tube.<br><br>
-
Resuspend the cells with 1 mL of Cacl2 at 0,1M + 15% glycerol for each 50 mL tube.<br/>
+
 
-
Split the total 4 mL into 40 tubes containing each 100 µL of concentrated cell solution. This step should be executed fast enough and on ice.<br/></p>
+
<b>. Wash membrane<br></b>
 +
Add 600 μL Buffer A4 (wash buffer) previously supplemented with ethanol. Centrifuge ar 11 000 x g for 1 minute. Discard flow-through and place the column back into an empty collection tube.<br>
 +
 
 +
<b>. Dry membrane<br></b>
 +
Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.<br><br>
 +
 
 +
<b>. Elution de l'ADN<br></b>
 +
Place the column in a 1,5 mL and add 50 μL de Buffer AE (elution buffer). Incubate at room temperature and centrifuge at 11 000 x g for 1 minute.<br>
 +
Measure the concentration with nanodrop, then stock the tubes at -20°C<br>
<script type='text/javascript'>writeFooter()</script>
<script type='text/javascript'>writeFooter()</script>
</html>
</html>

Revision as of 14:25, 30 July 2013

Plasmid purification

Protocole from Macherey-Nagel plasmid purification notebook

. Récupération des bactéries
Set saturated E.coli LM culture into 2 mL tubes. Centrifuge at 11 000 x g for 30 secondes. Discard as much as supernatant as possible.

.Cell lysis
Add 250 μL Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette.
Add 250 μL Buffer A2 (lysis buffer). Mix gently by inverting the tube 6 - 8 times. Incubate at room temperature until lysate appears clear.
Add 300 μL Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .
. Clarification of lysate
Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.

.Bind ADN
 Place a NucleoSpin Plasmid Column in a Collection Tube of 2 mL et and set the supernatant from the last step. Centrifuge at 11 000 x g for 1 minute. Discard flow-through and place the column back into the collection tube.

. Wash membrane
 Add 600 μL Buffer A4 (wash buffer) previously supplemented with ethanol. Centrifuge ar 11 000 x g for 1 minute. Discard flow-through and place the column back into an empty collection tube.
. Dry membrane
 Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.

. Elution de l'ADN
 Place the column in a 1,5 mL and add 50 μL de Buffer AE (elution buffer). Incubate at room temperature and centrifuge at 11 000 x g for 1 minute.
Measure the concentration with nanodrop, then stock the tubes at -20°C

Retrieved from "http://2013.igem.org/Team:Evry/Protocols/01"