Feed Back, Oct 2006. Santhosh Chelian of UC Davis. Fresh rice leaves or tree needles were powdered or pulverized with a MiniBeadbeater using several 2.5 mm or 3.2 mm chrome steel beads in a microvial. The capped vial contents were immersed 1/2 way into liq. N2 and and then quickly inserted into a MBB. No liq. N2 is added to the vial. Pulverize for one minute. Refreeze and repeat for another 1/2 min, if necessary. 40-60 mg of plant material was used in each well. (BIOSPEC NOTE: Do not use 6.35 mm steel beads for dry milling in our MBB machines, as they will blast right through the vials! Glass beads of the same 6.35 mm size, however, are okay. For MiniBeadbeater-96 users, the same cryogenic protocol works using 12 strips of 1.2 ml micro dilution tubes (96 well format, USA Scientific, #1212-8000). Also, consider using the solid aluminum vial holder, Cat. No. 702ALU, chilled to liq. N2 temperatures, with an array of individual microvials in the MBB-96).
After cryo-pulverizing the above tissue, DNA or RNA extraction solution was added to the vial and the mixture was "beadbeat" for an additional 1-3 minutes at room temperature. Nucleic acids were recovered in high yield. (BIOSPEC NOTE: Cryo-pulverized tissue, gently extracted with DNA extraction solution with no further physical homogenization, yields very large molecular weight DNA.)
Freeze-dried leaf tissue can also be dry ground. In this case, cryo-temperatures are not needed during grinding. Use a few 3.5mm or two 6.35mm glass beads per well or vial and grind for 3-5 minutes.
Preliminary information suggests that plant and animal tissue can be disrupted more quickly with sharp-edged material rather than smooth beads. Furthermore, it may not be necessary to prechop larger sized tissue into small pieces before "beadbeating". If you want to check this out, we now stock three sizes of Silicon Carbide sharp particles.
Feedback, Mar 2006. James Brien of Health & Science, University in Portland, OR. Brien reports that several whole organs from mice, cut into 3-4 pieces, were completely homogenized using 1 mm dia. SiC sharp particles. His objective was to measure virus load using a plaque assay. He found that the usual prechopping of the tissue to 1mm cross-section pieces was not necessary. Using a dye permeability test, he estimated that better than 99% of the cells were lysed after 2 minutes of shaking in a MiniBeadbeater-96. Lysis in both 2 ml and 7 ml vials was tested. One half of the vial volume was filled with the sharp particles. Controls using similar loads of 1 mm dia. spherical glass beads gave poorer results.
- Coating beads is a waste of time. The coating will be quickly removed during shaking.
- Most bead media is reusable. Wash with lab detergent (not acid!) and rinse well (see Bead Washing link below).
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