Team:Evry/Protocols/03
From 2013.igem.org
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<h2> Principle </h2> | <h2> Principle </h2> | ||
+ | <p>The aim of the purification step is to recover the plasmid produced by the bacteria (cloning strain).</p> | ||
<h2> Preparation </h2> | <h2> Preparation </h2> | ||
Revision as of 11:28, 26 August 2013
Plasmid purification
Principle
The aim of the purification step is to recover the plasmid produced by the bacteria (cloning strain).
Preparation
Protocole from Macherey-Nagel plasmid purification notebook1. Cell culture
Cultivate cells in LB medium overnight.
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.
Test
Before the storage of the tubes at -20°C, measure the concentration with nanodrop.
260/280 ratio indicate ...
260/230 ratio indicate ...
If the concentration is between 30 and 50 ng/μL,
If the concentration is below 30 ng/μL or if 260/280 ratio or 260/230 ratio is respectively..., make another purification.