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Bead Beating: A Primer (continued)

ANIMAL

Soft Animal Tissue Homogenization: Liver/Brain

Animal tissues, such as blood, liver, brain and thymus, are easily homogenized by bead beating. The small amount of connective tissue allows for these soft tissues to be processed very efficiently. Soft animal tissues can be homogenized with a buffer or cryogenically, the latter method being more useful for isolating high molecular weight DNA. Samples less than 100 mg can be processed in disruption tubes with 1.4-3.0 mm zirconium beads. Smaller samples can also be homogenized in polypropylene deep well plates with one 5/32” grinding ball or 6 mm satellite per well.

Larger samples are best homogenized in grinding vials. Processing up to 200 mg of animal tissue is best done in a 4 ml polycarbonate vial with one 5/16” or 3/8” stainless steel grinding ball. Up to two grams of animal tissue can be homogenized in a 15 ml polycarbonate vial and two 7/16” stainless steel grinding balls. In both instances, grinding can be done with buffer, or cryogenically without buffer. Cryogenic grinding is best done using Cryoblocks to prevent excessive melt back of the sample. Polyethylene vials should be used instead of polycarbonate when using extraction buffers containing phenol and/or chloroform. Zirconium oxide grinding satellites are chemically resistant and can be used with corrosive extraction buffers.

Procedures

1. Soft animal tissue can be processed in many different formats, though square deep well plates should be avoided.  When bead beating with homogenization buffer, do not to overfill tubes with sample and buffer as homogenate can be very thick.  For cryogenic grinding, omit buffer during the processing.  Buffers can be added after grinding, or the sample can be left frozen (and archived) until needed.
2. Prepare samples according to the following table:
Disruption Tubes (2 ml) Deep Well Plates (Round wells) 4 ml Vials 15 ml Vials
Sample Mass 20 mg 20 mg 100-200 mg Up to 2 gm
Buffer Volume 600 µl 200 µl 1 ml 6 ml
Grinding Balls 8 x 2.8 mm 1 x 5/32” 1 x 3/8” 2 x 7/16”
Processing Time 2 minutes 2 minutes 2-3 minutes 3-5 minutes
Bead Beater Speed High High High High
3. Process the samples as stated in the above table. Processing can be divided into short bursts followed by a cooling period for heat sensitive samples.
4. Sample homogenization can be monitored visually. A homogenate with a smooth appearance and lack of solids is a desirable end product. Disruption efficiency can be measured using a marker enzyme, such as lactate dehydrogenase, which is liberated from cells during processing.
5. Lysate can be stored or processed as needed.

Suggested Products

Balls: 2.8 mm Stainless Steel (GBSS 089-100-07), 5/32’’ Stainless Steel (GBSS 156-5000-01), 3/8’’ Stainless Steel (GBSS 375-1000-02), 7/16’’ Stainless Steel (GBSS 437-1000-03)
Pre-filled Tubes: 2.8 mm Stainless Steel (PFSS 2800-50-20)
Disruption Tube: 2 ml Skirted Disruption Tubes (PPDT 02-500-13)
Vial Sets: 24 Well PC Vial Set (PCVS 04-240-03), 15 ml Polycarbonate Vial Sets (PCVS 15-050-22)
Homogenization Vessel: See Appendix A
Homogenizers: Cryogenic grinding requires a GenoGrinder® or Mini G™ (Appendix A)
Cryogenic Option: Cryoblocks (4 ml and 15 ml)