MoBio PowerSoil®DNA Isolation Kit Protocol for Water samples 

 

Items not provided in the extraction kit:

 

• DNA decontaminating solution (DNAaway, 10% bleach, etc.)

• Microcentrifuge (10,000 x g)

• Pipettors (50 ul - 500 ul)

• Vortex-Genieâ 2 Vortex (MO BIO Catalog# 13111-V or 13111-V-220)

• Vortex Adapter (MO BIO Catalog # 13000-V1-24)

• 100% ethanol (for the PowerVac™ Manifold protocol only)

 

 Items included in the extraction kit :

 

 

 

 

 

 

 

 

 

 

 

 

Before you start:

 

Clean all surfaces and pipettors to remove DNA

Make sure the 2 ml PowerBead Tubes rotate freely in your centrifuge without rubbing.

Shake to mix Solution C4 before use.

 

Protocol

 

Please wear gloves at all times.

 

1. Centrifuge the water at 10,000 x g for 2 minutes to pellet the cells and then resuspend this cell pellet in the Bead Solution

 

2. To the PowerBead Tubes provided, add cell pellet from water sample

 

3. Gently vortex to mix.

 

4. Check Solution C1. If Solution C1 is precipitated, heat solution to 60 °C until dissolved before use.

 

5. Add 60 ul of Solution C1 and invert several times or vortex briefly.

 

6. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder for the vortex (MO BIO Catalog# 13000-V1) or secure tubes horizontally on a flat-bed vortex pad with tape. Vortex at maximum speed for 10 minutes.

Note: If you are using the 24 place Vortex Adapter for more than 12 preps, increase the vortex time by 5-10 minutes.

 

7. Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing. Centrifuge tubes at 10,000 x g for 30 seconds at room temperature.

CAUTION: Be sure not to exceed 10,000 x g or tubes may break.

 

8. Transfer the supernatant to a clean 2 ml Collection Tube (provided).

Note: Expect between 400 to 500 ul of supernatant. Supernatant may still contain some soil particles.

 

9. Add 250 ul of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5 minutes.

 

10. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.

 

11. Avoiding the pellet, transfer up to, but no more than, 600 ul of supernatant to a clean 2 ml Collection Tube (provided).

 

12. Add 200 ul of Solution C3 and vortex briefly. Incubate at 4°C for 5 minutes.

 

13. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.

 

14. Avoiding the pellet, transfer up to, but no more than, 750 ul of supernatant into a clean 2 ml Collection Tube (provided).

 

15. Shake to mix Solution C4 before use. Add 1200 ul of Solution C4 to the supernatant and vortex for 5 seconds.

 

16. Load approximately 675 ml onto a Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Discard the flow through and add an additional 675 ml of supernatant to the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Load the remaining supernatant onto the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature.

Note: A total of three loads for each sample processed are required.

 

17. Add 500 ul of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g.

 

18. Discard the flow through.

 

19. Centrifuge again at room temperature for 1 minute at 10,000 x g.

 

20. Carefully place spin filter in a clean 2 ml Collection Tube (provided). Avoid splashing any Solution C5 onto the Spin Filter.

 

21. Add 100 ul of Solution C6 to the center of the white filter membrane. Alternatively, sterile DNA-Free PCR Grade Water may be used for elution from the silica Spin Filter membrane at this step (MO BIO Catalog# 17000-10).

 

22. Centrifuge at room temperature for 30 seconds at 10,000 x g.

 

23. Discard the Spin Filter. The DNA in the tube is now ready for any downstream application.

 

No further steps are required.

 

 

We recommend storing DNA frozen (-20° to -80°C).

Solution C6 contains no EDTA.

 

To concentrate the DNA see the Hints & Troubleshooting Guide HERE.