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-	<b>1. Cell culture</b><br>	+	<b>1. Cell culture</b><br><br>
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	<b>2. Cell harvesting</b><br>		<b>2. Cell harvesting</b><br>

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Revision as of 16:06, 30 July 2013

Plasmid purification

Protocole from Macherey-Nagel plasmid purification notebook

1. Cell culture

2. Cell harvesting
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.

3. 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 .

4. Lysate clarification
Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.

5. DNA Binding
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.

6. Membrane washing
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.
7. Dry membrane
Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.

8. DNA Elution
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