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Team:Evry/Protocols/01
From 2013.igem.org
(Difference between revisions)
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<i>Protocole from Macherey-Nagel plasmid purification notebook</i><br><br> | <i>Protocole from Macherey-Nagel plasmid purification notebook</i><br><br> | ||
| - | <b>. | + | |
| + | <b>1. Cell culture</b><br> | ||
| + | |||
| + | <b>2. Cell harvesting</b><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> | 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> | ||
| - | <b>.Cell lysis</b> <br> | + | <b>3. Cell lysis</b> <br> |
Add 250 μL Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette. | Add 250 μL Buffer A1 (resuspension buffer). Resuspend the cells with a vortex or a pipette. | ||
<br> | <br> | ||
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Add 300 μL Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .<br><br> | Add 300 μL Buffer A3 (neutralisation buffer). Mix thoroughly by inverting the tube 6 - 8 times .<br><br> | ||
| - | <b>. | + | <b>4. Lysate clarification</b><br> |
Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.<br><br> | Centrifuge at 11 000 x g for 5 minutes . Repeat this step until supernatant is not clear.<br><br> | ||
| - | <b>. | + | <b>5. DNA Binding<br></b> |
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> | 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> | ||
| - | <b>. | + | <b>6. Membrane washing<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> | 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> | + | <b>7. Dry membrane<br></b> |
Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.<br><br> | Centrifuge at 11 000 x g for 2 minutes and discard the collection tube.<br><br> | ||
| - | <b>. Elution | + | <b>8. DNA Elution<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> | 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> | Measure the concentration with nanodrop, then stock the tubes at -20°C<br> | ||
Revision as of 16:05, 30 July 2013
Plasmid purification
Protocole from Macherey-Nagel plasmid purification notebook1. 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
