What would YOU do with a centrifuge?

[Positron]

Hi All.

I recently acquired a Sorvall GLC-2B centrifuge. It pulls a force of nearly 5,000G's on its contents @ 6,000RPM.

See: my centrifuge (http://websites.labx.com/rankin/pics/1046.jpg).

What would you do with it? I need some ideas...biochemistry experiments, organic chemistry, etc.

I am not a biochemist by any means, but I heard that it's not too incredibly hard to extract DNA via one of these. If I recall (and believe that I read it in a paper from a link off of this forum) it involved crushing animal tissue (or human inside-the-mouth tissue?) under liquid nitrogen, in a mortar and pestle. Separate out something with solvents...put the filtrate in a centrifuge for 30 minutes...extract a layer...dye it....Wa-laa: Recognizable patterns of main proteins A/T/G/C. (wow, and I actually remembered what those were, from many years ago in High School.)

Anyhow, the procedure went something like that.

[Dr. M]

I hope you got a good price.

As a Biochemist by trade, I can tell you that you need a good bit of brushing up on your knowledge of DNA and its constituents; "extract a layer and dye it" and "main proteins A/T/C/G" makes me think your biology is more than a little rusty.

First, as a safety note, inspect the rotor armature, the bucket pivots, and the buckets/other sample carriers that come with the machine; if possible, get an idea of the hours on the rotor and contact Sorvall for proper de-rating instructions for the rotor housing and its sample carriers. Just because the dial goes to 6k doesn't mean the thing will actually handle it under all configurations. Most of this information (at least relevant to when the equipment was all-new) should be in the operator's manual, but centrifuge companies' tech service departments are generally VERY helpful when it comes to this sort of thing; liability's a bitch, and rotor failure - under ANY circumstance - is catastrophic. Google search for "centrifuge rotor failure" and plenty of links - for example, from Purdue and MIT - will come up, including imagery that should make you think twice about irresponsible use.

Furthermore, the number one rule of centrifuge safety is balance. The second rule of centrifuge safety is balance. You'll know an imbalance when you first feel it - centrifuges should be smooooooth as butter when they fire up; every 'fuge has a "sweet spot" at which the whole piece resonates during spin-up, not matter how balanced it may be... however, this will be a momentary "shudder" very small in magnitude, and will disappear as rapidly as it appeared. Keeping your hand on the 'fuge as it spins up, and the other hand on the emergency stop, will allow you to minimize the potential for catastrophe. NEVER walk away from a centrifuge until it's spun up; walking two or three steps away is fine if you're nervous at first, but be damn sure to use your eyes and ears to sense danger. You're messing with a LOT of kinetic energy, and it's best not to let it get even an iota out of line.

Now, to answer your question about preparing DNA, I am sorry to disappoint you but your centrifuge will not suffice for this purpose. Cell pellets, crude extract partial clarification, separating crystalline substance from supernatant, etc. it will do very well; spinning out DNA it will not.

VERY briefly, standard techniques for DNA purification revolve around lysis of nucleotide-rich tissue, sedimentation of cellular debris (via centrifuge), digestion of RNA with an enzyme known as RNAse A, extraction of the mixture first with buffer-saturated phenol, decanting the aqueous supernatant, extracting with chloroform (to remove trace phenol in the now mostly pure mixture), decanting once again, then precipitating the DNA with ethanol or isopropanol at low temperature.

The procedure varies in its minutia, of course, depending on whether one wants to purify whole genomic DNA (e.g. human chromosomes) or plasmid DNA (small circular DNA entities used for genetic engineering of bacteria and other unicellular organisms). The great thing about isolating plasmid DNA is that, using alkaline lysis at room temperature followed by neutralization with a cold acetic acid/potassium acetate solution, one may denature the DNA in the mixture and rapidly cause the significantly larger genomic fraction to coagulate and spin out with the cellular debris; the smaller, usually supercoiled plasmid DNA remains in solution and is purified as I've mentioned above.

The unfortunate part is that final precipitation of the DNA creates such small nanoparticles in suspension that centrifugation at forces of minimally 10,000 x G (or 10,000 RCF if you like the modern nomenclature) are required to obtain any sort of appreciable yield. Your model simply won't put up that kind of force; however, if you have more in the budget I highly recommend the Eppendorf 5415C microcentrifuge (or a related member of the same family of 'fuges). If you can afford a large benchtop Sorvall, I imagine the Eppy won't be much of a stretch.

Finally, once you have purified DNA, there's really not all that much you can do with it unless you have access to the reagents and technical expertise; if you're thinking of framing someone for murder, and you can get a cheek swab or a blood sample, it's far better (and easier!) to just plant the initial sample on-scene rather than a purified extract. If it's just for fun, then I can give you detailed procedures for many things that'd probably tickle your creativity bone!

In all honesty, the best use I can think of off the top of my head for a non biochemically trained home user (related to the life sciences) would be growing large cultures of brewing or baker's yeast from a purchased starter-pack, and spinning the cultures down for freezing in order to always have a cheap and abundant supply ready at-hand.

If you have any more questions about this sort of thing, it's my bread and butter... don't hesitate to PM me if you don't want to post openly!

[BlackFalcoN]

What I would do if I were you ?
Put it on eBay and wait for señor El BlackFalcone to show up to make a very modest bid on this nice piece of equipment. ;)

Seriously though, something that would be very possible with this machine is to extract toxic proteins/ lectins from various plant materials. (Abrus Precatorius, Ricinus Communis, etc)

I won't go into detail on the exact procedures involved in this thread, since it has been posted in the more spefic threads (+ I don't want to encourage you to do so, if you don't have any real desire to perform these specific extractions - just because you COULD does not mean you SHOULD) but search the forum for lectin extraction; and research some of the later quoted sources by myself and other posters on centrifugal extraction of lectins.

[Charles Owlen Picket]

A centrifuge is commonly used as a filter of sorts. It pulls one material from another - generally fluids and mostly on a small scale. It is used when time is at issue and also has specific glass vessels in which to place the samples. The advantages are speed and containment. Disadvantages are small scale (or individual scale at least) and the loading, unloading process. I think of it as a filter. There are some neat things you could do with it but a lot depends on the strength of the machine and it's overall design.

[Positron]

Thanks for your replies. Sorry it took so long for me to get back, I should have mentioned that I'd be away for a while.

Dr. M, that's absolutely fascinating. I knew that it was more complicated than the hand-waving description that I first posted, but admittedly I didn't realize it was THAT complicated. It does however, sound like something that will be within my reach in the years to come.

Thanks for mentioning the hazards that the centrifuge can pose. Indeed, I am intimately familiar with high-energy mechanical things, and I kept a damn close eye on it (even though safety glasses) when I initially spun it up. It whirrs pleasantly :)

Obtaining the centrifuge was probably the coolest swap that I've ever done. I swapped it for a very nice Solid State Tesla Coil that I built back in '02 :)

I won't go into detail on the exact procedures involved in this thread, since it has been posted in the more spefic threads (+ I don't want to encourage you to do so, if you don't have any real desire to perform these specific extractions - just because you COULD does not mean you SHOULD)

I've done a lot of things just because I could, but I see what you're saying. I love the unconventional and will try just about anything...though I'll do it carefully, slowly, and with great control. Separating the proteins sounds intriguing, and I've actually been reading up on that subject quite a lot over the last year or so. I haven't put anything to use...yet! :D

Thanks, those were the replies I was hoping for.