Bead Beating: A Primer (continued)
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Leaf tissue can easily be homogenized by bead beating. Leaf punches, made with a common paper or Harris punch, are commonly used for genetic analysis. Small samples up to 50 mg (about five 6 mm leaf punches) can be processed in polypropylene deep well plates with one 5/32” stainless steel or zirconium oxide grinding ball per well; these samples can also be processed in disruption tubes with 1.0- 3.0 mm zirconium beads or 2.8 mm stainless steel balls. Denser leaf tissue with heavy cuticle will require larger beads and balls.
Samples with masses above 100 mg are best homogenized in grinding vials. Processing of up to 200 mg of leaf tissue is best done in 4 ml polycarbonate vials with one 5/16” or 3/8” grinding ball. Up to a gram of leaf tissue can be homogenized in 15 ml polycarbonate vials with two 7/16” stainless steel grinding balls. Both 4 ml and 15 ml vials can be purchased as Vial Sets, meaning that they are packaged in a rack with stainless steel grinding balls and caps. Zirconium oxide is an alternative to stainless steel grinding balls, which may react when used with corrosive buffers.
Leaf tissue is often a source of genomic DNA, and the method chosen to grind it will significantly affect the size of the isolated fragments. Bead beating in solution will generate smaller DNA fragments than dry or cryogenic grinding. Smaller fragments are adequate for use in many applications, such as PCR, but if high molecular weight DNA is required, then leaf tissue should be either lyophilized prior to grinding or homogenized cryogenically. Cryoblocks, which keep samples frozen and dissipate heat during processing (see Cryogenic Homogenization above), are typically used for cryogenic grinding.
|1.||One leaf punch is approximately 10 mg (depending upon the plant). Multiple punches can be placed in one tube or larger pieces of leaf can be weighed and added to vessels. Grinding balls are most effective, though larger size beads can be used if available.|
|2.||If dry grinding lyophilized leaf, omit the buffer. For cryogenic grinding, use either 4 ml or 15 ml polycarbonate vials and omit the buffer. Deep well plates and disruption tubes will often crack if used for cryogenic grinding. Prepare samples according to the following table:|
|Disruption Tubes (2 ml)||Deep Well Plates (square wells)||4 ml Vials||15 ml Vials|
|Sample Size||50 mg||50 mg||200 mg||Up to 1 gm|
|Buffer Volume||600 µl||600 µl||2 ml||6 ml|
|Grinding Balls||8 x 2.8 mm||1 x 5/32”||1 x 3/8”||2 x 7/16”|
|Processing Time||1 minute||1 minute||2 minutes||2-5 minutes|
|Bead Beater Speed||2/3 speed||2/3 speed||2/3 speed||2/3 speed|
|3.||Process the samples as stated in the table. Processing can be divided into short bursts followed by a cooling period for heat sensitive samples not processed cryogenically.|
|4.||Check the homogenate following processing. Typically a well processed sample will appear as a smooth green liquid. If large pieces of leaf tissue are present, repeat the processing.|
|5.||Sample can be stored or processed as needed.|
|Balls:||2.8 mm Stainless Steel Grinding Balls (GBSS 089-1000-07), 5/32” Stainless Steel Grinding Balls (GBSS 156-5000-01), 3/8” Stainless Steel Grinding Balls (GBSS 375-1000-02), 7/16” Stainless Steel Grinding Balls (GBSS 412-1000-03)|
|Pre-filled Tubes:||3.0 mm Zirconium (PFAW 3000-50-17), 2.8 mm Stainless Steel (PFSS 2800-50-20)|
|Disruption Tubes:||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:||See Appendix A|