TRNL Test Samples - Extraction

Owned by Shannon Harris
Last updated: Jul 09, 2021
2 min read

  • 32 samples (half plant/half stool) submitted for test extractions.

  • Samples weighed out and placed in PowerBead Pro Tube prior to submission.

Extraction Protocol:

  1. Add 800uL of solution CD1 using the 1250uL pipette. Vortex briefly to mix.

  2. Homogenize samples thoroughly using Vortex Adapter for 1.5-2mL tubes. Vortex at max speed for 10 minutes. (If using more than a 12 prep adapter, increase vortex time by 5-10 mins.)

  3. Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min.

  4. Transfer the supernatant to a clean 2mL microcentrifuge tube (provided in kit). NOTE: Expected supernatant should be around 500-600uL. Some soil particles may remain in the supernatant.

  5. Add 200uL of solution CD2 using the 1250uL pipette and vortex for 5 seconds.

  6. Centrifuge at 15,000 x g for 1 min. Avoiding the pellet, transfer up to 700uL of supernatant to a clean 2mL microcentrifuge tube (provided in kit). NOTE: Expect 500-600uL and avoid pellet for best quality and yields.

  7. Add 600uL of solution CD3 using the 1250uL pipette and vortex for 5 seconds.

  8. Load 650uL of the lysate onto an MB Spin Column using the 1250uL pipette and centrifuge at 15,000 x g for 1 min.

  9. Discard the flow-through and repeat step 8 with the remaining lysate.

  10. Carefully place the MB Spin Column into a clean 2mL collection tube and avoid getting any flow-through on the MB Spin Column during transfer.

  11. Add 500uL of solution EA to the MB Spin Column using the 1250uL pipette. Centrifuge at 15,000 x g for 1 min. (EA is a wash buffer for protein and non-aqueous contamination removal).

  12. Discard the flow-through and place the MB Spin Column back into the same 2mL collection tube.

  13. Add 500uL of solution C5 to the MB Spin Column using the 1250uL pipette. Centrifuge at 15,000 x g for 1 min. (C5 is an ethanol-based wash solution for residual salt, humic acid, and other contamination removal).

  14. Discard the flow-through and place the MB Spin Column into a new 2mL collection tube.

  15. Centrifuge at up to 16,000 x g for 2min (dry spin for residual ethanol removal). Carefully place the MB Spin Column into a new 1.5mL elution tube.

  16. Add 100uL of solution C6 to the center of the white filter membrane using 1250uL pipette.

  17. Centrifuge at 15,000 x g for 1 min. Discard the MB Spin Column and close elution tube with final DNA product.