8/11/2019 DNA Rumen Extraction

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Extraction of DNA/RNA from rumen contents or fecesDr. 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 vesselbe sparged with CO2. 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 buffersDNA: 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 (DNAor 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 (

8/11/2019 DNA Rumen Extraction

2/2

Extraction of DNA/RNA from rumen contents or fecesDr. 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