Extraction of DNA/RNA from rumen contents or feces

Dr. Paul Weimer

Last update: 07/2013 (K. Jewell)
Go as deep as possible into the rumen (avoid surface layers). For fluids, place a large
handful of material into the center of four sheets of tightly-woven cheesecloth, twist it into a sort
of a bag, and squeeze it hard over a funnel leading to a sterile tube. If possible, have the vessel
be sparged with CO
2
. For solids, shove the squeezed material into a second vessel. You need
~30 mL of liquids and ~50 mL of solids. Transport on wet ice and freeze at -80C.
Extraction buffers
DNA: 100 mM tris/HCl, 10 mM EDTA, 0.15 M NaCl, pH 8.0
RNA: 50 mM sodium acetate, 10 mM EDTA, pH 5.1, use glacial acetic acid to adjust the pH
Solids (and hard feces)
1. Thaw the samples at room temperature in a water bath prior to blending. This takes ~45 min.
2. Blend each sample for 1 min at a low speed with 80 mL of ice-cold extraction buffer (DNA
or RNA) that was used to rinse the contents of the collection tubes. Add 5-10 mL of
additional buffer as needed for smooth blending.
3. Transfer the homogenate to centrifuge tubes (depends on available centrifuge). Rinse the
blender with a small amount (<30 mL) of cold extraction buffer and add to tubes.
4. Centrifuge at 500 g for 15 min at 4C. This removes large contaminating particles while
keeping cells in suspension.
5. Pour the washes into fresh centrifuge bottles through 4 layers of cheese cloth OR use a little
glass wool at the bottom of a funnel. Glass wool is difficult to use, but wetting it with buffer
makes it easier to handle.
6. Add 30 mL chilled extraction buffer to the remaining solids, vortex, centrifuge, and filter
(use the same filter) as above to capture remaining fiber-adherent cells.
7. To collect the cells, centrifuge at 8,000 g for 25 minutes (or 5,000 g for 1 hr) at 4C.
Balance tubes with cold extraction buffer.
8. Label chloroform-resistant 1.5 mL Eppendorf tubes (6 per sample) and bead-beating tubes
(one per sample) by adding: 0.5 g of 0.1 mm zirconium beads, 50 L of 20% SDS, and 700
L equilibrated cold phenol (pH 8.0 for DNA, pH <5.0 for RNA) to 2.0 mL screw-cap tubes.
9. Resuspend and combine cell pellets in cold extraction buffer (DNA or RNA).
10. Process as for liquids, starting with adding 1 mL of cell suspension in each screw-cap tube
(Step 5).
Liquids (and soft feces)
1. Thaw the samples at room temperature in a water bath. This takes 45 min - 1 hr.
2. To collect the cells, centrifuge thawed tubes at 8,000 g for 25 minutes (or 5,000 g for 1 hr)
at 4C. Balance tubes with cold extraction buffer.
3. Label chloroform-resistant 1.5 mL Eppendorf tubes (6 per sample) and bead-beating tubes
(one per sample) by adding: 0.5 g of 0.1 mm zirconium beads, 50 L of 20% SDS, and 700
L equilibrated cold phenol (pH 8.0 for DNA, pH <5.0 for RNA) to 2.0 mL screw-cap tubes.
4. Resuspend the cells in 2 - 4 mL of cold extraction buffer (DNA or RNA).
5. Put 1 mL of cell suspension in each screw-cap tube. Use a cut-off tip if the particles clog a
normal tip.
6. Bead-beat at 4C for 2 minutes.
7. Heat in a water bath at 60C for 10 minutes.
8. Bead-beat again at 4C for 2 minutes.
Extraction of DNA/RNA from rumen contents or feces
Dr. Paul Weimer
Last update: 07/2013 (K. Jewell)
9. Centrifuge for 10 minutes in a table-top centrifuge at maximum rpm (~14,000 for most
machines). This can be done at RT, but if the rotor gets warm then the phenol will fail to
clear or separate as well. Try to do all centrifugations at 4C.
10. While centrifuging, add 400 L of phenol (again, pH 8.0 for DNA or <5.0 for RNA) to each
new tube (TUBE 1). Keep all reagents cold.
11. Remove about 850 L of the top (aqueous) layer CAREFULLY to each 1.5 mL Eppendorf
tube; this is the aqueous phase containing DNA. Leave any solid material behind. It is better
to lose sample than carry over this debris!
12. Vortex vigorously and centrifuge rpm for 10 min.
13. While centrifuging, add 400 L of phenol and 100 L of extraction buffer to each new tube
(TUBE 2) to keep up total volume.
14. Remove about 700 L of the top (aqueous) layer and add to each tube.
15. Vortex vigorously and centrifuge at max rpm for 10 min.
16. While centrifuging, add 400 L of phenol and 120 - 150 L of extraction buffer to each new
tube (TUBE 3).
17. Remove about 650 L of the top (aqueous) layer and add to each tube. Sometimes this step is
not necessary if the aqueous phase is completely clear. You will always need this step with
rumen-based samples.
18. Vortex vigorously and centrifuge at max rpm for 5 minutes.
19. While centrifuging, add 500 L of phenol/chloroform (pH ~8.0 for DNA or <5.0 for RNA)
and 120 - 150 L of extraction buffer to each new tube (TUBE 4).
20. Remove about 650 L of the top (aqueous) layer and add to each tube.
21. Vortex vigorously and centrifuge at max rpm for 5 minutes.
22. While centrifuging, add 500 L of phenol/chloroform (pH ~8.0 for DNA or <5.0 for RNA)
and 120 - 150 L of extraction buffer to each new tube (TUBE 5).
23. Remove about 650 L of the top (aqueous) layer and add to each tube.
24. Vortex vigorously and centrifuge at max rpm for 5 minutes.
25. While centrifuging, add 1/10 volume (~50 L) 3 M Na acetate and 0.6 total volumes (~300
L) of isopropanol to each tube (TUBE 6).
This is a possible stopping point. Store at -20C.
26. Remove 450 - 500 L of the top (aqueous) layer and add to each tube.
27. Vortex vigorously and centrifuge at max rpm and 4C for 20 minutes.
28. Pour off or pipette out the isopropanol.
29. Add 1 mL of 70% ethanol to each tube without disturbing the pellet.
30. Centrifuge at max rpm for 1 min and remove the ethanol.
31. Add 500 L of 100% ethanol and centrifuge at max rpm for 1 min.
32. Remove the ethanol, then add 500 L of 100% ethanol and centrifuge at max rpm for 1 min.
This is a possible stopping point. Store at -20C.
33. Remove ethanol. Use a P200 pipette to remove as much as possible.
34. Centrifuge at max rpm for 3 min and use a pipette to remove remaining ethanol.
35. Air dry for ~20 minutes. Make certain all ethanol is gone.
36. Resuspend the pellet of DNA in 50-100 L TE or molecular-grade water for DNA (10 mM
tris/HCl, 1 mM EDTA, pH 8.0) or RNAse-free water for RNA and store at -20C.