Bead Beating: A Primer (continued)
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DNA Isolation from Animal Tissues
The separation of DNA from other biomolecules in lysates is traditionally accomplished by exploiting DNA solubility characteristics. In animal tissues, DNA can be released from the cell by bead beating either cryogenically or in solution. Homogenization buffers usually contain Tris buffer, EDTA, SDS, and Proteinase K. SDS can foam extensively during bead beating and can be added after sample processing. Though most methods used to isolate DNA employ silica spin columns (usually part of a kit), the protocol below uses a traditional method that relies upon spooling the DNA onto glass rods or Pasteur pipettes.
Homogenization Buffer: 50 mM Tris, pH 9, 100 mM EDTA, 200 mM NaCl.
Other Solutions: 10% SDS, 10 mg/ml Proteinase K in water, TE buffer (10 mM Tris, pH 8, 1 mM EDTA), phenol/chloroform/isoamyl alcohol (in a ratio of 25:24:1) stored under TE buffer (DO NOT GET THIS SOLUTION ON YOUR SKIN AS IT BURNS!), 3M sodium acetate, 95% ethanol (cold)
|1.||Depending upon the use of the DNA, samples may best be homogenized cryogenically and then suspended in homogenization buffer. If high molecular weight DNA is needed, homogenize the samples cryogenically. Alternatively, samples can be homogenized in the buffer and then supplemented with SDS and Proteinase K.|
Cryogenic Grinding – Following cryogenic bead beating, add 8 volumes of homogenization buffer as compared to the mass of the sample (i.e., if the sample is 100 mg, add 0.8 ml). Add 1 volume of each 10% SDS and 10 mg/ml Proteinase K. Mix and incubate at 55°C for 1 hr.
Solution Grinding – Add sample and buffer to a vessel along with beads/balls. Process the sample as described. Open the vessel and add 1/8 volume (original buffer volume) of each 10% SDS and 10 mg/ml Proteinase K. Mix and place at 55°C for 1 hr.
|2.||Add an equal volume of phenol/chloroform/isoamyl alcohol to the homogenate. Cap the vessels and mix by gently inverting. Centrifuge to separate the phases. The aqueous phase containing the DNA is on the top. Carefully remove the aqueous phase with a polypropylene pipette tip and transfer to a clean tube.|
|3.||Add 1/10 volume of 3 M sodium acetate and mix. Using ice-cold ethanol, carefully layer two (2) volumes of the ethanol over the DNA solution. A cloudy phase will form on the interface of the ethanol and aqueous phase. This is the where the DNA is spooled onto glass.|
|4.||Using a clean Pasteur pipette, insert the tip into the cloudy interface and slowly twirl. DNA will stick to the glass. Remove the spooled DNA from the tube and wash off the Pasteur pipette with a small volume of TE buffer (minimize this volume). If the DNA did not spool onto the glass, then continue on to step 5.|
|5.||If the DNA was excessively sheared during homogenizing, it may not spool. Thus continuing, the tube with ethanol can be mixed and placed in a 20°C freezer for 1 hr to allow DNA to precipitate. Centrifuge the DNA solution at 10,000 x g for 20 minutes. Decant and wash the pellet with 70% ethanol to remove residual salt. Air dry the pellet, but do not allow the pellet to become dry (the goal is to remove extraneous alcohol).|
|6.||Resuspend the pellet in a small volume of TE buffer (e.g., 20 µl). Store at -20°C until needed.|