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Methods for Homogenizing Soybean

There are many ways to prepare soy beans for protein, DNA, and RNA isolation. In these protocols, the focus is on soybean. Homogenization of the vegetative portions of soy, i.e., leaves, stems, and roots, can be homogenized using the standard techniques found in the application note for "General Plant Homogenization". For seeds, below are several protocols using high throughput homogenizers.

 

Protocols

Mixer Mill Preparation

Mixer mills such as the Geno/Grinder and HT Homogenizer are necessary when processing a large number of soybean samples. Soybeans can be homogenized using bead beating in several ways: dry, wet, or rehydrated. For beans which have been air dried or lyophilized, grinding can be accomplished without use of extraction buffers. Alternatively, extraction buffers can be used to homogenize dried beans, but this is less efficient than dry grinding as sample volumes must be smaller to allow for the volume of the buffer. Rehydrating beans in water or a buffer can be done prior to homogenization. This makes the grinding process easier by making the sample less resilient.

  1. Place bean in 4 ml vials. For dry grinding or grinding using aqueous buffers, use polycarbonate vials.  If buffers are used that have phenol and/or chloroform, use polyethylene vials.  Add a 3/8" stainless steel grinding ball to the vial. Cap the vial and place into a rack or a vial set.

  2. For dry grinding, skip this step.  For homogenization with buffer, add about 500 µl of buffer if homogenizing a rehydrated or wet soybean.

  3. Place the rack into the mixer mill and process on high for 5 minutes (Geno/Grinder or HT Homogenizer).  If following the processing the beans are not completely pulverized, repeat the process.

  4. Balls can be removed from the Vial Set using the 24 Pin Magnet Ball Dropper or with magnet tips.

  5. For wet grinding, transfer supernatant with the appropriate sized pipette to a new tube if desired.

Pooled or Batch Processing

  1. Up to 15 or more dry soybeans can be processed using a 15 ml vial sets. These larger vials make use of two 7/16" stainless steel grinding balls.

  2. The process is the same as single seed dry grinding described above.  One important point is that larger sample sizes usually take more time to process.

Note: Mixer mill bead beating does generated heat and may cause denaturing of certain enzymes and protein. Heat can be mitigated by submersing the tubes and cryogenic rack into liquid nitrogen before homogenization. Cryoblocks are available and act as cold sinks for cryogenic grinding.

References

Comparison of Methods for DNA Extraction from SoyBeans.  OPS Diagnostics Application Notes.