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maradydd ([personal profile] maradydd) wrote2009-02-07 02:42 am
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Drinking straw electrophoresis!

Gel electrophoresis is one of the most versatile, widely used tools in a microbiologist's or geneticist's toolbox. It's used for separating out DNA, RNA or protein molecules (that you presumably isolated in a previous step of your experiment) based on their molecular weight, so that you can analyze the molecules, clone them, amplify them with PCR, sequence them, lots of different things.

Electrophoresis does require some equipment to perform -- an inner tray which holds the gel, an outer tray which holds a "running buffer" solution (which keeps things cool and keeps pH stable), electrodes, and a power supply (50V-150V is pretty common). You can buy a gel box from a commercial supplier, though they're not cheap, and a fancy power supply will set you back even more; Bio-Rad has some nice ones, but they run to the thousands of dollars.

Happily, there are solutions for the biohacker on a budget. The University of Utah's genetics department has full specs for how to build your own gel box for about $25 in parts (not counting the power supply, which will run you about $50). The main components are clear acrylic and acrylic cement, which I purchased and had cut to size at TAP Plastics -- they also do mail order. My partner-in-science Tito Jankowski built one too, and did some test runs with food colouring which enabled him to separate the individual dyes which make up different colours. (The molecules in food colouring are pretty small, which is why the bands in Tito's video are a little smeary. He used agarose -- an edible, seaweed-derived polymer which you can find on the shelf in any Asian grocery store, also sold as "vegan gelatin" -- as his gel, and agarose is better suited to larger molecules like DNA. But it's definitely a proof of concept!)

Still, electrophoresis using large rectangular gels has some drawbacks. It's a bit messy, and in order to recover the particular band of DNA you want, you have to slice it out of the gel with a razor blade or something similar. Cleaning up the equipment is also a bit of a pain. If you're using acrylamide or polyacrylamide (common for protein electrophoresis), you need to find a safe way to get the used gel out of the gel carrier and dispose of it properly. Also, while DNA electrophoresis is run horizontally, protein electrophoresis is done vertically, so that means two different pieces of equipment.

This was a recent topic of discussion on the DIYbio mailing list. Ben Lipkowitz wondered whether it would be possible to use a narrow, rigid tube to contain the gel, rather than a big carrier. This would allow for the use of less buffer and lower voltage, since a physically smaller amount of gel is a smaller resistor.

Well, what's a narrow rigid tube that's easy for anyone to acquire? A clear drinking straw! Paper clips make for appropriately sized electrodes, and since a drinking straw is rigid, it can be used in either the horizontal or the vertical orientation. For extra bonus points, when you're ready to cut a band out of the gel, no need for mucking around with razor blades -- just take a (sterile) pair of scissors, snip snip, and you're done! Plus, disposal is extra simple, even with polyacrylamide -- just dispose of the entire straw, gel and all, properly.

Tito Jankowski tried this out, using a single 9V battery as a power supply, and after some debugging, it worked beautifully. (He also used alligator clips as electrodes, and they worked just fine.) We're calling these "keiki gels" because they're so small and cute -- and so simple, even a little kid can do them.

This is crowdsourced science at its very finest. Behold the power of collaboration!

Tito's keiki gels!


ETA: Tito wrote a protocol, doo dah, doo dah

Re: !/2 filled straw....horizontal..cutting..

[identity profile] maradydd.livejournal.com 2009-02-09 06:17 am (UTC)(link)
It seems to me that the 'thumb' of the seam ripper would leave a trail of displaced molecules behind it when making the cut.

Even if the straw were only half-full longitudinally?

The boba straw idea is a good one. A quick Google search doesn't turn up much in the way of purely clear straws, but I wonder if translucent orange or translucent blue would work as a light filter?

Re: !/2 filled straw....horizontal..cutting..

[identity profile] keith-m043.livejournal.com 2009-02-09 06:54 am (UTC)(link)
Even if the straw were only half-full longitudinally?

yes that should work, (I beg pardon for not reading all comments before posting, I did not catch the half-full part)

The first boba straw I saw was clear with thin blue longwise stripes. I would expect that a lot of them would be clear as a lot of ppl like to watch the tapioca ascend.

[identity profile] kragen.livejournal.com 2009-02-09 04:45 pm (UTC)(link)
It will depend on how consistent the gel is; just as two carbon-composition resistors made the same size and the same way may have resistances that are off by a few percent, two gels made the same way might have resistivities that are off by a few percent.

Buffers are necessary!

(Anonymous) 2009-02-09 08:37 pm (UTC)(link)
TBE is a buffer! Salt water is not. During electrophoresis you will produce H+ and OH- ions, ie the ph will change. This is why you need a buffer. Ph changes are not good for biomolecules. Using salt will produce chlorine, a pretty strong oxidizer and not good to breathe. Oxidixers are not good if your goal is keeping protiens and nucleic acids intact. Also TBE is used for nucleic acids as the boric acid forms a complex with them, improving the resolution. No ideas off the top of my head for a cheap substitute.

[identity profile] cathshaffer.livejournal.com 2009-02-10 05:57 pm (UTC)(link)
You are doing a lot of reinvention of the wheel.

You can make a perfectly fine slab agarose gel by taping together pieces of glass or plastic. I don't think you're going to get usable results with DNA using agar. Those dye bands are very huge and smeary, and DNA is not going to migrate any tighter. The size of the molecule affects the rate of migration through the gel, but it doesn't make the band itself any tighter. The size of the band is a function of the percentage agarose and the quality of the casting. You might get usable results by running tube gels in parallel, but they would need to be cast from the same batch because small variations in agarose concentration will have a big effect on the results.

You would need a good number of drinking straws do sequence a whole genome. (LOL) I believe Celera actually used capillary electrophopresis (shotgun method) for the human genome project.

[identity profile] maradydd.livejournal.com 2009-02-10 06:01 pm (UTC)(link)
Just to clarify, I've already made my own rectangular gel box from acrylic. So has Tito. We wanted to try this to see how well it would work. :)

FWIW, dye bands are huge and smeary in a rectangular gel as well; there's a video of them migrating on his blog. So I am pretty sure that DNA bands in a straw will come out cleaner than the dye bands as well.

We'll be doing some more tests with lambda DNA soon, and I'll post an update when that happens.

And yeah, based on comments upthread, it should be pretty easy to cast hundreds if not upward of a thousand straws at the same time.

[identity profile] cathshaffer.livejournal.com 2009-02-10 06:04 pm (UTC)(link)
When you say rectangular gel, you're talking about your home made rectangular gel poured with agar?

[identity profile] maradydd.livejournal.com 2009-02-10 06:09 pm (UTC)(link)
Yep. I linked the design docs in the original post -- it's the one from the University of Utah.

[identity profile] cathshaffer.livejournal.com 2009-02-10 06:11 pm (UTC)(link)
Okay, so your bands are smeary in both. The probably means it's the agar. If your DNA bands are smeary as well, you'll probably want to upgrade to agarose.

[identity profile] maradydd.livejournal.com 2009-02-10 06:15 pm (UTC)(link)
Yep, proper agarose is on the list too. (There's got to be a way to separate out agarose from the other polymers in agar-agar, but that probably falls into the "too much hassle, just buy it from a supplier" category.)

[identity profile] cathshaffer.livejournal.com 2009-02-10 08:20 pm (UTC)(link)
Here's an easy protocol for home electrophoresis:

http://www.nexusresearchgroup.com/fun_science/electrophoresis.htm

There's also a nice picture of a gel run with food coloring dyes.

I think you would really enjoy taking some classes in biochemistry and genetics. The theoretical grounding would help you make better decisions about your techniques and goals, even if you do continue working in your home.

[identity profile] maradydd.livejournal.com 2009-02-10 08:53 pm (UTC)(link)
I understand that you're trying to help, but I can't help but feel a little insulted by your last remark there. I don't pretend to be an expert biologist by any stretch of the imagination, but I did spend three years as a bioinformaticist at IDT, have had a couple of bioinformatics publications, and spent most of last year working in a proteomics group.

That said, I'm proud to be an amateur. My ultimate goals are exploration, learning, and lowering the barrier to entry for other people. I'm also fundamentally an autodidact. Some of my projects will fail. Some of them will succeed up to a point and get no further. And, frankly, I'm okay with that. Hell, Real Scientists(tm) have that problem too. (Being a Real Scientist(tm) in another field, I feel I can say that with authority. ;) )

[identity profile] cathshaffer.livejournal.com 2009-02-10 09:45 pm (UTC)(link)
Well, I'm certainly not going to apologize for suggesting you enroll in classes in a subject that you obviously love quite a bit. Bioinformatics is a really distinct field from biochemistry/molecular biology. I have B.S. and M.S. degrees in Biochemistry and have taught introductory lecture and lab classes in the field. I've also done research in academic and industrial laboratories, and I now earn my living by freelance writing in the field of biotechnology. It's hard enough to a successful cloning experiment, say, when you have all of the resources of a multi-billion dollar company at your fingertips, including a high tech sequencing core facility, every type of instrument and freezers full of all of the reagents and supplies you can use. The reality of research is such that it fails...over and over again, even when you're doing everything right, and you end up tweaking extremely fiddly details in order to finally get where you want to go. Having the tools to do the work is not the real hurdle.

I'm not trying to insult you, but I will say that you will be more successful if you have a theoretical grounding in the science. So you are an autodidact. What have you read? Maybe I can give you some recommendations.

Great idea

(Anonymous) 2009-02-11 03:51 am (UTC)(link)
Hi Meredith!

Wow you are a fascinating person!

Thank you for posting about the Drinking straw electrophoresis. What a wonderful idea!

I came across an article about your work with yogurt. Very impressive and I’m hopeful I can do something similar in a home setting. I do plant tissue culture and am interested in the genetic aspect of it also. I would like to combine the two interests. I do this as a hobby. I love science too:-)

I found two favorite websites today! Yours and DIYbio.
Frank

(Anonymous) 2009-02-25 07:09 pm (UTC)(link)
Ha ha. You all are not old enough to remember that the original gels WERE tube gels, before folks invented the slab gel! So you are reinventing the wheel. But hey, nowadays nobody makes their own ANYTHING in the lab- they buy it all premade. Back in my day you had to make your own gel boxes- now the DIYbio folks are going back to that. Next we'll be purifying EcoR1 from E. coli again....

[identity profile] maradydd.livejournal.com 2009-02-25 07:20 pm (UTC)(link)
Given that buying EcoR1 from a supplier can be a real pain in the ass for a person not affiliated with a commercial or university lab, we may very well be purifying it from E. coli again sometime quite soon, actually.

[personal profile] hattifattener 2009-03-09 06:40 am (UTC)(link)
This would allow for the use of less buffer and lower voltage, since a physically smaller amount of gel is a smaller resistor.
That seems backwards to me. Isn't it the electric field (voltage gradient) that determines how fast stuff migrates? If you think of the straw as just being a slice out of a slab gel (blah blah symmetry argument blah), then you'll want the voltage gradient (V/cm) to be the same, but the current draw will be proportionately smaller than for a slab.

OTOH, my mol-bio sweetie says you can run a gel pretty darn slow before diffusion starts making it blurry, so maybe it doesn't matter that much. Apply whatever voltage you've got and wait for the bands to separate.

(and, oh, yeah: cool idea!)
ext_6297: cool dog, cooldog, cool, dog, fonz (Default)

this is totally epic

[identity profile] cyanfox.livejournal.com 2009-03-30 10:24 am (UTC)(link)
this is toally epic

i'm totally going to have to show this to all of my friends and freak out my boss at work XD

Please, help me, brothers!!!

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Amateur research

(Anonymous) 2009-05-19 02:48 pm (UTC)(link)
If you don't include my childhood I have been involved with amateur and professional research and development for over 30 years. I have designed and built a great many things in the basement I was told could not be done, yet often I got excellent results. I am a engineer with a background in instrumentation. When someone tells me something can not be done I become very curious, I want to know why. I have found they will usually give me clues as to how to make the system work better. Oddly enough I would have to say the key to my success has more to do with my abilities to build a model of a system and modeling the data from a system in a computer than my design expertise. Although building a model from theory has its advantages (especially at the start), when you are breaking new ground it will usually get in your way as you start making attempts to improve the system. What I see is an opprotunity for dirt cheap automation.

straw design

(Anonymous) 2009-05-19 05:43 pm (UTC)(link)
It appears to me it would be rather easy to make a tube with a different density of jell on each end. Put them in the right direction and it would be a piece of cake to separate molecules and end up with a storage package.

Lab power supply

(Anonymous) 2009-05-20 08:05 pm (UTC)(link)
I question the need for a lab quality power supply. With the use of a computer controlled sound board and a garage sale stereo amp it would be a piece of cake to make a pulsed power supply, the voltages are easily within reach. Second with the use of a analogue game controller you could easily add computer control to the system. Of course a bit of programming would be required.

media design

(Anonymous) 2009-05-20 08:20 pm (UTC)(link)
As soon as you consider the possibility of manufacturing a straw with more than one media density then the possibility of minimizing the edge effects that may cause an issue can be adjusted. The rheology of the basic media makes this fairly simple, a matter of controlling temperature and extrusion rate. A bit of knowledge of basic mechanical systems and a trip to the hardware store with 20 buck and a little recycling should handle it.

Separating Agar into componets

(Anonymous) 2009-05-20 10:44 pm (UTC)(link)
Since one component of the agar is not electrically neutral it should be possible to make a separator making use of a capacitor design. An AL cookie sheets, some insulating sheet material (plastic wrap), and a way to charge the sheet should do the trick.
Cover the cookie sheet with plastic wrap, apply charge to cookie sheet, hold over a container at an angle TBD, pour solution over sheet into container. The part you want should end up in the container with correct charge on the cookie sheet.

Straw electrophoresis

(Anonymous) 2010-01-02 02:11 am (UTC)(link)
Thank you so much for this very cool idea! My teenage daughter and I love science. She's won several state and regional fairs, presented her national project at the Smithsonian, and has several novel ideas for cell replication studies in the works. But most of this "should" be done in a stocked, certified university lab. Ever tried to convince a PhD to take on a 14 year old kid's research project in their lab? Brilliance and innovation can't hold up against the threat of liability. So..she converted a spare bathroom into a lab, along with HEPA filtration, borrowed a microscope, and used her allowance to order culture dishes. But the PCR and gel were beyond reach. Now with a couple of candy thermometers, a soap dish, and drinking straws...she'll be back in business! Not to mention a microcentrifuge made out of a salad spinner (yup...it really works). Thanks to people like you for your awesome ideas!!

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