Extraction of the Plasmid DNA from Bacteria without NucleoSpin® Plasmid

The experiment is performed with A1, A2 and A3 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. Removal of protein:

Recover the supernatant of step 3 and add 800µl of Phenol (1 Volume of supernatant for 1 Volume of phenol) Centrifuge for 5 min at 11.000*g. Two phases appear : an organic phase and an aqueous phase. Phenol denatures proteins in the sample and denatured proteins stay in the organic phase whereas the aqueous phase contains DNA. Recover the supernatant very carrefully.

5. Isolation of DNA:

Add between 1600µl and 2000µl of cold ethanol at 100%. Add sodium acetate to obtain 0.3M finally. Ethanol and sodium acetate creat a precipitate because of the caracteristic hydrophobic of ethanol. Then sodium acetate creat link between DNA molecule because of the presence of some positive ions. Put the tube at -20°C The formation of this precipitate may long more than 30min.

6. Wash and isolation of DNA:

Centrifuge for 10min at 11.000*g and 4°C Discard the supernatant Add 1ml of Cold ethanol 70% Centrifuge for 10min at 11.000*g and 4°C once again Discard the supernatant Dry the pellet

Add 30 to 50µl of buffer of water milliQ

7. Analysis:

Check the presence of DNA and determine its concentration by Electrophoresis.