Farmer Lab Detailed Protocol

From Protists

Please wear gloves at all times



1. Get tube of concentrated log-phase cells from -80°C cell box. Allow to defrost at room temperature (RT).

  • This step concentrates and pellets the microbial cells. In some cases it may take longer to completely pellet the cells. It is important to pellet the cells completely and remove all the culture media in this step.

2. Resuspend the cell pellet in 300 ul of MicroBead Solution and gently vortex to mix. Transfer resuspended cells to MicroBead Tube.

  • The MicroBead Solution contains salts and a buffer which stabilizes and homogeneously disperses the microbial cells prior to lysis.

3. Add 50 u­l of Solution MD1 to the MicroBead Tube.

Solution MD1 contains SDS and other disruption agents required for cell lysis. In addition to aiding in cell lysis, SDS is an anionic detergent that breaks down fatty acids and lipids associated with the cell membrane of several organisms. If it gets cold, it will precipitate. Heating at 60°C will dissolve the SDS and will not harm the SDS or the other disruption agents. In addition, Solution MD1 can be used while it is still warm.

4. Optional: To increase yields, to minimize DNA shearing, or for difficult cells, see Alternative lysis methods in the "Additional Information" section before continuing. Note: Our lab puts the tubes into the dry/water bath (preheated) at 65°C for 10min.

  • This optional step can lead to better performance in some cases. We recommend using only one of these methods for any individual prep.

5. Secure bead tubes horizontally using the MO BIO Vortex Adapter tube holder for the vortex (Catalog No. 13000-V1-24) or secure tubes horizontally on a flat-bed vortex pad with tape. Vortex at maximum speed (actually only ~6.5) for 10min. Be careful attachment doesn't fly off! (See "Alternative lysis method" for less DNA shearing).

  • This step creates the combined chemical/ mechanical lysis conditions required to release desired nucleic acids from microbial cells. Many cell types will not lyse without this chemically enhanced bead beating process. The vortex action is typically all that is required, however, more robust bead beaters may also be used. In most cases the times may be shorter with other devices but you may run the risk of increased DNA shearing. This process is compatible with fast prep machines.

6. Make sure the 2 ml MicroBead Tubes rotate freely in the centrifuge without rubbing. Centrifuge the tubes at 10,000g for 30s at RT. CAUTION: Be sure not to exceed 10,000g or tubes may break.

  • The cell debris is sent to the bottom of the tube while DNA is remains in the supernatant.

7. Transfer the supernatant (i.e. top liquid) to a clean 2 ml Collection Tube (provided).

8. NOTE: Expect 275 to 350 u­l of supernatant. The volume to expect will vary depending on the size of the original cell pellet from step 1.

9. Add 100 u­l of Solution MD2, to the supernatant. Vortex 5s. Then incubate at 4°C for 5min.

10. Centrifuge the tubes at RT for 1min at 10,000g.

  • Solution MD2 contains a reagent to precipitate non-DNA organic and inorganic material including cell debris and proteins. It is important to remove contaminating organic and inorganic matter that may reduce DNA purity and inhibit downstream DNA applications.

11. Avoiding the pellet, transfer the entire volume of supernatant to a clean 2 ml Collection Tube (provided). Expect approximately 450 u­l in volume.

  • The pellet at this point contains non-DNA organic and inorganic materials, including cell debris and proteins. For the best DNA quality and yield, avoid transferring any of the pellet.

12. Add 900 u­l of Solution MD3 to the supernatant and vortex 5s.

  • Solution MD3 is a highly concentrated salt solution. It sets up the high salt condition necessary to bind DNA to the Spin Filter membrane in the following step.

13. Load about 700 u­l into the Spin Filter and centrifuge at 10,000g for 30s at RT. Discard the flow through, add the remaining supernatant to the Spin Filter, and centrifuge at 10,000g for 30s at RT. NOTE: A total of 2 to 3 loads for each sample processed are required. Discard all flow through liquid in MoBio DNA Extraction Kit Waste container under hood (satellite accumulation area).

  • DNA is selectively bound to the silica membrane in the Spin Filter device. Contaminants pass through the filter membrane, leaving only the DNA bound to the membrane.

14. Add 300 u­l of Solution MD4 and centrifuge at RT for 30s at 10,000g.

  • Solution MD4 is an ethanol based wash solution used to further clean the DNA that is bound to the silica filter membrane in the Spin Filter. This wash solution removes residues of salt, and other contaminants while allowing the DNA to stay bound to the silica membrane.

15. Discard the flow through in the aforementioned waste container.

  • This flow through is waste containing ethanol wash solution and contaminants that did not bind to the silica Spin Filter membrane.

16. Centrifuge at RT for 1min at 10,000g.

  • This step removes residual Solution MD4 (ethanol wash solution). It is critical to remove all traces of wash solution because it can interfere with down stream DNA applications.

17. Being careful not to splash liquid on the spin filter basket, place Spin Filter in a new 2 ml Collection Tube (provided). Label as follows on top and side of tube.

Image:HowToLabelTubesSmall.jpg

  • It is important to avoid any traces of the ethanol based wash solution.

18. Add 50 u­l of Solution MD5 to the center of the white filter membrane.

  • Placing the Solution MD5 (elution buffer) in the center of the small white membrane will make sure the entire membrane is wetted. This will result in more efficient release of bound DNA

19. Centrifuge at RT for 30s at 10,000g.

  • As the Solution MD5 (elution buffer) passes through the silica membrane, DNA is released, and it flows through the membrane, and into the Collection Tube. The DNA is released because it can only bind to the silica Spin Filter membrane in the presence of salt. Solution MD5 is 10mM Tris pH 8 and does not contain salt.

20. Discard Spin Filter. DNA in the tube is now ready for any downstream application. Put scotch tape over labels on tube prior to storing. No further processing steps are required.


21. Put tube into DNA box in the -80°C and update DNA.xls sheet with the tube(s) information.



  • We recommend storing DNA frozen (-20°C). Solution MD5 contains no EDTA.
  • Thank you for choosing the UltraClean(trademarked) Microbial DNA Isolation Kit.



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