Q&A on the use of the MiniBeadbeater

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Selected Technical Questions and Answers on the MBB:

----- Original Message -----

From: John Adamczyk

Sent: Monday, May 12, 2003 10:40 AM

Subject: Mini-beadBeater-96

Tim:

We have the 8-er in my lab but are looking to upgrade. Will the 96-er accept 2.0 ml tubes with the 6 mm (1/4") stainless steel ball bearings, or will it only homogenize with glass beads? You can use stainless steel beads. But, most people use much less expensive chrome steel beads of the same size. This works well for most plant material being homogenized in liquid extraction media. Softer, animal tissue usually does not require steel (or tungsten carbide) beads. By using less dense glass, zirconia-silica or zirconia beads, the price of the beads are cheap enough to be considered "disposable" - thus saving you time and money. Note that 6 mm steel beads are too large when "dry milling" air-dried or freeze-dried plant material (i.e., no liquid is present) . Due to the very high shaking energies of the MBB series, the plastic microtubes will be breached. Rather, use microvials filled 1/3 - 1/2 full of 3.2 mm chrome steel beads. See our web page for a list of bead media to choose from and further guidance in the proper selection of size and density. ........PS: John's reply: - a couple of "1/4 inch steel beads worked great in the 2 ml deepwell microplates. Plant tissue was homogenized in extraction buffer in only one minute".

What is the minimum amount of tissue that one can use with the 96-er to conduct PCR? Some use as little as 10 mg of fresh tissue. Depends somewhat on the tissue used. The maximum, by the way, is about 400 mg. Using this larger amount one should pre-chopped the sample into pieces about 1mm in cross-section. Use more than that and efficiency of cell disruption drops.

Do you still have to fill the tubes with the maximum about of solution (e.g. about 1.0 ml)? Yes, otherwise you get foaming and that degrades the efficiency of cell disruption. And, in your case, the cell disruption should be done in nucleic acid extraction media such as chloroform-phenol or some appropriate DNA extraction "kit" solution.

Also, I'll need prices from two of your competitors in order to justify since I work for the government, etc. It is our view that there are no competitors for the Minibeadbeater-96 as none come close to the required energy input required for efficient, fast cell disruption. Our closest "competitor" is probably the Retsch MM300. See our detailed comparison of the Retsch versus the MBB on our web site.

-----Original Message----------------------------------------------
From: Tim Hopkins
To: Isaac Fruchey
Sent: 5/27/2003 6:08 PM
Subject: Re: Optimal cell disruption in the MiniBeadbeater series and their different shaking speeds

There are several factors to take into consideration in getting good cell disruption using the bead mill technique. First and foremost is the bead size (0.1 mm, in your specific case) and type of bead material (zirconia-silica). The vial should be filled up at least 1/2 full with beads and the remaining volume in the vial filled with spores and the breaking buffer or media. Try to eliminate most of the trapped air phase when sealing the tube. Time of cell disruption should not exceed 5 minutes - usually 3 min does the job. Shaking speed should be at the maximum setting of the machine.

The shaking speeds of the three MiniBeadbeaters we manufacture all differ. Their posted values are no-load speeds. The MBB-1 and MBB-8 use brush motors. Fully loaded with samples, they run somewhat slower than the posted speed. The MBB-96 uses an induction motor and does not run slower under load.

Shaking speed is not the only machine variable to achieve good cell breakage. The shaking pattern (straight back and forth, figure eight, etc), the shaking direction within the vial (top to bottom vs side to side), the shaking position of the vial itself (horizontal vs vertical), the shaking distance (3/4", 1 1/4", etc), and the abruptness of reversal in shaking direction all interact to give successful cell disruption.

BOTTOM LINE. It turns out, perhaps fortuitously, that the unique combination of machine variables of each of the MBB models deliver similar end results -- i.e., one does not have to modify an established cell breakage protocol when going from one MBB model to the another.

-----Original Message--------------------------------------------

To: Beth Hughes

Subject: Re: Integrity of the 96-well Microplate Format During Shaking

Possible cross contamination between wells of a microplate is a valid concern. Microplates are sealed with flexible "mats" (an array of little stoppers) or with an adhesive film. BioSpec's testing of laboratory of mats and films from different manufactures, while not comprehensive, has revealed big differences in performance. To date we find that Axygen Scientific makes deep well microplates and matching silicone sealing mats that perform well and we offer their brand on our web site. Mats made of other flexible polymers or that rely on adhesive-based attachment may not prevent leaking during intense shaking. We also note that the way microplates are clamped into the shaker is important to good sealing. The MBB-96 machine compresses the mat evenly over the entire top surface of the microplate. It is recommend that you test for possible well-to-well cross contamination when using plates and sealing methods of your choice to satisfy yourself that all is trustworthy. One easy way to do this is to place a drop of concentrated food color solution in alternate wells or in alternate rows of wells.

Should microplate integrity not meet your criteria, turn to time-tested, very secure microvials having an O-ring in the screw-cap. MiniBeadbeater-96's include a microvial holder that holds up to 45 of these microvials.

-----Original Message--------------------------------------------

Subject: Dry Grinding

A number of companies use the MBB series to grind air-dried or freeze-dried plant and animal tissue. This method works well with non-woody tissue and for tissue not having high lipid or oil content. Dry grinding is usually done at room temperature. Furthermore, Bruno Zelleni at Cornell University reports that dry grinding of lyophilized fungi gives very high MW DNA in good yields. Frozen fresh fungi ground in Liq. N2 with a mortar and pestle did not.

Use chrome steel (not expensive stainless) beads (Cat No. 11079132cs) in a straight walled 2 ml screw-cap vial. The vial should be 1/3 - 1/2 full of beads. Add the dry sample, perhaps crudely crushed ahead of time, and operate the machine at maximum speed for one-tenth to one minute.