Bead Beating: A Primer (continued)
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Animal tissues, such as muscle, heart and lung, require substantially greater force to homogenize than softer tissues because of significant amounts of connective tissue and microfilaments. Small whole animal samples, such as Drosophila and C. elegans, can be treated as small fibrous samples. Fibrous tissue can be homogenized with buffer or cryogenically, the latter being useful for high molecular weight DNA isolation.
Samples less than 50 mg can be processed in disruption tubes with 1.7-3.0 mm zirconium beads or small stainless steel balls. Small samples (10-50 mg) can also be homogenized in polypropylene deep well plates with one 5/32” grinding ball or 6 mm satellite per well.
Larger samples should be homogenized in grinding vials. Processing up to 200 mg of animal tissue is best done in a 4 ml polycarbonate vial with one 3/8” stainless steel grinding ball. Up to two grams of animal tissue can be homogenized in a 15 ml polycarbonate vial with two 7/16” stainless steel grinding balls. Polyethylene should be used over polycarbonate for samples that require organic solvents (phenol/chloroform buffers) that melt polycarbonate. Zirconium oxide grinding satellites are resistant to corrosive chemicals such as phenol and can be used with garnet shards to rip and cut the tissue.
|1.||Fibrous 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; it is important that the ball move freely during processing. For cryogenic grinding, omit buffer during the processing. Buffers can be added after grinding, or the sample can be left frozen 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||3-5 minutes||5-10 minutes|
|Bead Beater Speed||High||High||High||High|
|3.||Process the samples as stated in the table. For heat sensitive samples, the processing can be divided into short bursts followed by a cooling period.|
|4.||Sample homogenization can be monitored visually. A homogenate with a smooth appearance and lack of solids is a desirable end product. Using a microscope, muscle fibers should be highly fragmented, although complete disruption does not usually occur. Disruption efficiency can be measured using a marker enzyme, such as lactate dehydrogenase, which is liberated from cells during processing.|
|5.||Lysate can be processed as needed.|
|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)|