Preparation of Sample
Use 0.1 mm beads for bacteria, 0.5 mm beads for yeast, fungi and tissue culture cells and 1.0 mm beads for plant and animal tissue. Up to 400 mg wet wt of biomass can be disrupted per ml of extraction media. If you have solid tissue, pre-chop it with a single-edge razor blade or scalpel.**
** Tissue samples sizes over a few tens of milligrams should be prechopped into pieces less than 1 mm in cross-section. If your sample is already harvested and frozen, do not thaw. This is especially important if you are isolating nucleic acids. Rather, Cryopulverize the sample. For information on this method, see http://www.biospec.com/product/40/biopulverizer/.
Fill the screw-cap vial at least 1/2 full (1/2-3/4 is okay) with beads (BioSpec has a simple bead loader for 96-well microplates which makes loading a few plates easy and accurate, Cat. No. 702L. For loading numerous deep-well microplates, see Cat. No. 104L). Bead size and type should be matched to the biomaterial you are processing (see Beads for details). Then add your extraction media and cells, being sure to fill the microtubes or wells almost to the top. Exclude as much air from the microtubes or wells as is practical. We recommend using screw-cap microtubes with o-ring seals. All snap-top microtubes aerosol during shaking. Be sure there are no beads on the threads of the microtubes or on the lip area of the microplate(s) when sealing the microtube or microplate(s). If sealing microplates with a flexible mat or with adhesive film, firmly press the mat or film over the entire top surface of the microplate (BioSpec offers a manual, high leverage mat press which makes sealing easy and secure, Cat. No. IT-EP-R).
Some microplates and their matching mats or sealing films work better than others. You may want to try different sealing techniques and microplates and mats made by several different manufacturers to find one that delivers the integrity needed to prevent sample leakage from well to well during the shaking process in the MBB-96. To date, we find that microplate products P-DW-20-C (2 ml) and P-DW-11-C (1.1 ml) with silicone rubber sealing mats AM-2ML-RD made by Axygen Scientific (Union City, CA) pass our dye test. Please let us know if you find additional manufacturer brands that work, so that we can pass the information on to other users.
Operating the Mini-Beadbeater-96
1) Insert microtubes into the tube holder, if applicable. Distribute the tubes symmetrically. Place the rack of microtubes or microplate (if using 1 ml microplates you can stack two at a time) into the chamber holder, top side toward you. Screw down the two black plastic knobs until the top stainless steel retainer plate is in contact with the microtube caps or top of the microplate(s). Tighten knobs snug but not overly tight. Finally, screw down the white nylon wing nuts.
2) Turn on the 'on-off' switch on the front of the MBB-96 cabinet and select the cell disruption time (0-5 min). Typical cell disruption times are 3 minutes. If you are working with heat-sensitive material such as native proteins, consider pre-cooling your beads and sample, homogenizing for 1 minute, then removing the vials or plate and cooling in ice-water for 1 minute. Cycle for a total 'on' time of three minutes. Cooling is not need when extracting nucleic acids in nucleic acid isolation media.
3) Start the homogenization by pushing the button on the timer dial. The timer dial resets itself automatically at the end of the run.
You must operate the MBB-96 with the black plastic hood lowered over the chamber. This prevents the user from coming in contact with the shaker during operation and will trap anything should it break free. Do not by-pass the safety features of the lid.
Do not operate the MBB-96 in a cold room. Special electronics in the unit will prevent the machine from starting. Furthermore, cold air cannot effectively cool samples while beadbeating.