Genomic DNA extraction

Principle

The aim of the extraction step is to recover Escherichia coli genomic DNA.
The different step are use to throw other bacterial components off (proteins, cell wall, plasmids,etc).

Preparation

1. Cell culture
Cultivate cells in LB medium overnight.

2. Cell harvesting
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.

3. Cell lysis
Add 180 μL of Digestion Solution and 20 μL of Proteinase K Solution. Resuspend the cells thoroughly with a vortex or a pipette.
Incubate the tubes at 56°C while vortexing occassionally until the cells are completely lysed(~30 minutes).
Add 20 μL of RNase A solution, mix by vortexing and incubate the tubes for 10 minutes at room temperature.
Add 200 μL of Lysis Solution to the sample. Mix thoroughly by vortexing until a homogeneous mixture is obtained. (~15 secondes)
Add 400 μL of 50% ethanol and mix with a vortex or a pipette.

4. DNA Binding
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.
Discard the collection tube containing flow-through solution and place the column into a new 2 mL tube.

5. Membrane washing
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. Add 500 μL of Wash Buffer II (previously added with ethanol)to the purification column.

6. Dry membrane
Centrifuge at 16 000 x g for 3 minutes.

7. DNA Elution
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. Discard the purification column and store the purified DNA in TrisHCl at -20°C or use it immediatly.



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.