<i>Protocol adapted from Thermo Scientific Genomic extraction 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><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 8 000 x g for 5 minutes. Discard as much as supernatant as possible.<br><br><p>
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<p><b>3. Cell lysis</b> <br>
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Add 180 μL of Digestion Solution and 20 μL of Proteinase K Solution. Resuspend the cells thoroughly with a vortex or a pipette.
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Incubate the tubes at 56°C while vortexing occassionally until the cells are completely lysed(~30 minutes).<br>
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Add 20 μL of RNase A solution, mix by vortexing and incubate the tubes for 10 minutes at room temperature.<br>
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Add 200 μL of Lysis Solution to the sample. Mix thoroughly by vortexing until a homogeneous mixture is obtained. (~15 secondes)<br>
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Add 400 μL of 50% ethanol and mix with a vortex or a pipette.<br>
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<br>
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<p><b>4. DNA Binding<br></b>
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Transfer the lysate to a GeneJET Genomic DNA Purification Column inserted in a collection tube. Centrifuge the column at 12 000 x g for 1 minute.<br> Discard the collection tube containing flow-through solution and place the column into a new 2 mL tube.<br>
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<p><b>5. Membrane washing<br></b>
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Add 500 μL of Wash Buffer I (previously added with ethanol). Centrifuge at 1 600 x g. Discard the flow-through and place the purification column back into the collection tube.
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Add 500 μL of Wash Buffer II (previously added with ethanol)to the purification column.
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<br><br>
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<p><b>6. Dry membrane<br></b>
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Centrifuge at 16 000 x g for 3 minutes.
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<p><b>7. DNA Elution<br></b>
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Add 200 μL of Elution Buffer to the center of the purification column membrane to elute genomic DNA. Incubate for 2 minutes at room temperature and centrifuge at 8 000 x g for 1 minute.
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Discard the purification column and store the purified DNA in TrisHCl at -20°C or use it immediatly.
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