Extraction of the Plasmid DNA from Bacteria by using NucleoSpin Plasmid®

The experiment is performed with buffer solutions of NucleoSpin® Tissue, protocol 5.1 for bacteria.

1. Cultivate and harvest bacterial cells:

Take 2 ml of bacteria culture in a 2 ml microcentrifuge tube (eppendorf), prepare one sample. Centrifuge the culture for 30s at 11,000*g. Discard supernatant and remove as much of the liquid as possible.

2. Cell lysis:

Resuspend and dissolve the pellet in 250 µl Buffer A1 by pipetting up and down. Make sure no cell clumps remain before addition of Buffer A2. Add 250 µl Buffer A2. Mix gently by inverting the tube 6-8 times. Do not vortex to avoid shearing of genomic DNA. Incubate at room temperature for up to 5 min or until lysate appears clear. Add 300µl Buffer A3. Mix thoroughly by inverting the tube 6-8 times. Do not vortex to avoid shearing of genomic DNA!

3. Clarification of lysate:

Centrifuge for 5 min at 11.000*g at room temperature. Repeat this step in case the supernatant is not clear!

4. Bind DNA:

Place a NucleoSpin® Plasmid Column into a collection tube (2ml) and decant the supernatant from step 3 or pipette a maximum of 750µl of the supernatant onto the column. Centrifuge for 1 min at 11.000*g. Discard the flow-through and place the column back into the collection tube. Repeat this step to load the remaining lysate.

5. Wash silica membrane:

Add 600 µl Buffer A4 (supplemented with ethanol) to the column and centrifuge for 1 min at 11.000*g. Discard flow-through and place the NucleoSpin Column back into the empty collection tube.

6. Dry silica membrane:

Centrifuge the column for 2 min at 11.000*g and discard the collection tube.

7. Elute DNA:

Place the NucleoSpin Plasmid Column in a 1.5 ml microcentrifuge tube (not provided), and add 50 µl Buffer AE. Incubate at room temperature for 1 min. Centrifuge for 1 min at 11.000*g.

Discard the column.

8. Analysis:

Check the presence of DNA and determine its concentration by using a nanodrop spectrophotometer.