Team:Evry/Protocols/03

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<h1 align='center'> Protocole for Plasmid purification </h1>
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<p>For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA.<br/>
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<h1> Plasmid purification </h1>
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Let 30 minutes on ice.<br/>
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Let the cells at 42°C for exactly 50 seconds.<br/>
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Let 5 minutes on ice.<br/>
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Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.<br/>
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<h2> Goal </h2>
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<p>The aim of the plasmid purification step is to recover the plasmid produced by the bacteria (cloning strain).<br>
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The different step are use to throw other bacterial components off (proteins, cell wall, genomic DNA, etc).</p>
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<h2> Preparation </h2>
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<i>Protocol adapted from Macherey-Nagel plasmid purification notebook</i><br><br>
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<b><p>1. Cell culture</b><br>
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Cultivate cells in LB medium overnight.<br></p>
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<p><b>2. Cell harvesting</b><br>
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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><p>
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<p><b>3. Cell lysis</b> <br>
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Add 250 μL of Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette.
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<br>
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Add 250 μL of Buffer A2 (lysis buffer). Mix gently by inverting the tube 6 - 8 times. Incubate at room temperature until lysate appears clear.<br>
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Add 300 μL of Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .<br></p>
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<p><b>4. Lysate clarification</b><br>
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Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.<br></p>
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<p><b>5. DNA Binding<br></b>
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Place a NucleoSpin Plasmid Column in a Collection Tube of 2 mL 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></p>
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<p><b>6. Membrane washing<br></b>
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Add 600 μL of 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></p>
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<p><b>7. Dry membrane<br></b>
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Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.<br></p>
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<p><b>8. DNA Elution<br></b>
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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></p>
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<h2> Test </h2>
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<p>Before the storage of the tubes at -20°C, measure the concentration with nanodrop.</p>
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  <a title="Nanodrop" href="https://static.igem.org/mediawiki/2013/f/f0/Nanodrop.png">
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    <img alt="Nanodrop" src="https://static.igem.org/mediawiki/2013/f/f0/Nanodrop.png" class="Picture"/>
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    <b>Figure 1:</b> Figure 1: Nanodrop test after a plasmid purification<br/>
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<p>
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If the concentration is between 30 and 50 ng/μL, concentrate your DNA with SpeedVac concentrator or any other method.<br>
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If the concentration is below 30 ng/μL or if 260/280 ratio or 260/230 ratio is not correct, make another purification.<br><br>
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<i>260/280 ratio and 260/230 indicate the purity of DNA (or RNA). <br>
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Nucleic acids absorb at 260 nm while proteins and phenols absorb at 280 nm and carbohydrates at 230 and other contaminents at 230 nm.<br>
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For DNA, a 260/280 ratio around 1,8 is concider to be pure and ange 260/230 ratio must be between 2,0 and 2,2.</i><br></p>
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{{:Team:Evry/foot}}

Latest revision as of 13:25, 1 October 2013

Iron coli project

Plasmid purification

Goal

The aim of the plasmid purification step is to recover the plasmid produced by the bacteria (cloning strain).
The different step are use to throw other bacterial components off (proteins, cell wall, genomic DNA, etc).

Preparation

Protocol adapted from Macherey-Nagel plasmid purification notebook

1. 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 of Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette.
Add 250 μL of Buffer A2 (lysis buffer). Mix gently by inverting the tube 6 - 8 times. Incubate at room temperature until lysate appears clear.
Add 300 μL of 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 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 of 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.

Nanodrop
Figure 1: Figure 1: Nanodrop test after a plasmid purification

If the concentration is between 30 and 50 ng/μL, concentrate your DNA with SpeedVac concentrator or any other method.
If the concentration is below 30 ng/μL or if 260/280 ratio or 260/230 ratio is not correct, make another purification.

260/280 ratio and 260/230 indicate the purity of DNA (or RNA).
Nucleic acids absorb at 260 nm while proteins and phenols absorb at 280 nm and carbohydrates at 230 and other contaminents at 230 nm.
For DNA, a 260/280 ratio around 1,8 is concider to be pure and ange 260/230 ratio must be between 2,0 and 2,2.

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