who can give advice about larger sample number tissue crushing - methods/machines that can help w breaking of dozens of tissue samples (Jun/27/2008 )

Dear bioforum members,

Do get biomolecules from tissues (DNA/RNA/protein) I have previously used drills, freeze/thaw, syringes,.. All very time consuming and not scalable for large number of samples. The drill only allows me to do a sample per about 2-5min, freeze/thaw doesn't work well for tough tissue, syringes take even longer than drilling esp. because of cleaning.

That's why I'd like to ask around whether anybody here is using machines that facilitate the process of tissue disruption but allow for several samples to be crushed in parallel?

A quick search for possible tabletop machines yielded these two links. Any experience with these babies? Can the beads be recycled and solutions prepared or does this material have to be bought again and again?

- http://www.mpbio.com/product_info.php?cPat...ywords=fastprep
- http://www.precellys.com/en/applications/kits_and_protocols/

Hope to hear from you. Best,

j

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What about high-frequency ultrasonic energy with a sonifier? The emitter tips are easy to clean.
E.g. this supplier

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Hi hobgoblin,

Thanks for the idea. With these rod-like sound emitters you have to go tube by tube? That would take very loooong. Can you sonicate a batch of say 20 epis in a sonication water bath and disrupt weak tissue? What my colleagues tell me, I get the impression that especially the water bath sonication is not strong enough to disrupt tissue much. Still brooding

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You could try lysing the tissue samples in AquaGenomic at 65 *C for 4-5 hours and you don't even need a homogenizer.

1) Add 10 mg tissue to a microfuge tube containing 200 ul of AquaGenomic and ~50 ul volume of glass beads,

2) Incubate the samples (12 or 48, depending on the rotor capacity of your centrifuge) at 60-65 *C for 4-5 hours,

3) Vortex the samples vigorously for a minute to break up the tissue,

4) Centrifuge at 12000xg for 5 minutes to pellet the debris,

5) Take the supernatant and ethanol precipitate the DNA.

That's it. Simple, easy, hands-off, and avoiding DNA cross contamination between samples.

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Hi chessplayer,

Thanks for the tip. Sounds like a good way to do parallel extraction of DNA from many samples without too much hands-on time. What would you do to extract protein from many samples in parallel?

Best, j

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J,

We use AquaRNA for protein extraction. It extracts DNA/RNA/proteins from the same sample. It's a potent protein denaturant and extractant. If you are going after proteins only and not recovering DNA and RNA, the protocol will be simple.

1) Add 500 ul AquaRNA to 5 million cells, vortex to lyse the cells.
2) Add 500 ul isopropanol, vortex, and spin to pellet DNA/RNA.
3) Transfer the protein super to a new tube, add 4 ml acetone, vortex, and spin to pellet proteins.
4) Add 200 ul 1%SDS to suspend the protein pellet.
5) Transfer the protein suspension to a microfuge tube, heat to 90*C for 3 min to clear the solution, chill on ice for 15 min to form insoluble, spin to pellet the insoluble, transfer the purified protein supernatant to a new tube.

For processing multiple tissue samples, I notice you have the drill and you could homogenize the tissue samples in AquaRNA then proceed as above.

CP

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Ah, interesting. You adopted the RNA method to extract proteins. Thanks for the tip.

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