Extraction of the Genomic DNA from Bacteria by using NucleoSpin® Tissue

The experiment is performed with buffer solutions of NucleoSpin® Tissue, protocol 6.2 for bacteria.

1. Prepare sample:

Take 1 ml of bacteria culture in a 1.5 ml microcentrifuge tube (eppendorf), prepare one sample. Centrifuge the culture for 2 min at 13,200*g. Remove and discard supernatant.

2. Pre-lyse sample:

Resuspend and dissolve the pellet in 180 µl Lysis Buffer T1 by pipetting up and down. Add 25 µl Proteinase Buffer PB (Proteinase K). Agitate vigorously with vortex and incubate at 56°C for 1 h (until complete lysis). Vortex every 10 min during incubation. Heat up the Elution Buffer BE at 70°C.

3. Lyse sample:

Vortex the sample. Add 200 µl Lysis Buffer B3, Agitate vigorously with hand and incubate at 70°C for 10 min. If the insoluble particles are visible, centrifuge the samples for 5 min at 13,200*g and transfer the supernatant to a new 1.5 ml microcentrifuge tube. Discard the insoluble particles.

4. Adjust DNA binding conditions:

Add 210 µl ethanol 100% to the sample, stringy precipitates are visible. Agitate vigorously with hand.

5. Bind DNA:

Place a NucleoSpin® Tissue Column into a collection tube. Apply the sample to the column, make sure to load all of the precipitate. Centrifuge for 1 min at 13,200*g. Discard the flow-though and place the column back into the collection tube.

6. Wash silica membrane:

1st wash:

Add 500 µl Wash Buffer BW to the column and centrifuge for 1 min at 13,200*g. Discard the flow-though and place the column back into the collection tube.

2nd wash:

Add 600 µl Wash Buffer B5 to the column and centrifuge for 1 min at 13,200*g. Discard the flow-though and place the column back into the collection tube.

7. Dry silica membrane:

Centrifuge the column for 1 min at 13,200*g to remove the residual ethanol. Discard the flow-though.

8. Elute highly pure DNA:

Place the column into a 1.5 ml microcentrifuge tube, add 100 µl Elution Buffer BE (pre-warmed at 70°C). Incubate at room temperature for 1 min. Centrifuge for 1 min at 13,200*g.

Discard the column.

9. Analysis:

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