Keiki gels: Gel electrophoreis in a straw

[Tito Jankowski]

Hi everyone,

It works - with food coloring, at least. DNA is next.

My results:
http://titojankowski.com/

Coverage by Meredith Patterson:
http://maradydd.livejournal.com/417631.html

Tito

[Josh Perfetto]

Hi Tito,

Do you know how much consistency you are getting in the separation between
straws? It sounds like there might be some issues here since you said one
of the straws didn't seem to separate much. From the pictures it looks like
the alligator clips are just sitting in the buffer, is that the case? If so
you could probably easily get a much better result simply by running a
taught bare wire through the buffer at each end of the gel box, and
attaching the alligator clips to these wires somewhere outside of the
buffer, as this would create a more uniform field.

At any rate great result and thanks for posting!

-Josh

[Meredith L. Patterson]

And it's been boingboinged.
http://www.boingboing.net/2009/02/06/crowdsourced-science.html

Go Tito!

--mlp

[Tito Jankowski]

Hi everyone,

Here's a basic protocol - give it a shot. Does it work for you? I used
9v batteries, straws, and agar powder from the Chinese store.

http://openwetware.org/wiki/DIYbio:Notebook/Keiki_Gels

Tito

[Nick Taylor]

This is so cool I can hardly stand it. It's a perfect example of what I find incredibly interesting at the moment.

n

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[Steve Dickie]

I used to be a taxonomist (in a former life) now I'm a high school
physics teacher. I have a couple quick questions. Hopefully they
aren't too ignorant, I haven't done any DNA work in about ten years.
First I want to say this is totally awesome and I will be passing this
along to other teachers I know.

Do all the straws run at the same rate? If not, how do I know which
band I want?

How do you stain for DNA in a straw? All the protocols I used (again,
10 years ago) involved staining the gel after a run.

Thanks,
Steve

On Feb 7, 3:29 am, Tito Jankowski <titojankow...@gmail.com> wrote:
> Hi everyone,
>
> Here's a basic protocol - give it a shot. Does it work for you? I used  
> 9v batteries, straws, and agar powder from the Chinese store.
>
> http://openwetware.org/wiki/DIYbio:Notebook/Keiki_Gels
>
> Tito
>
> On Feb 6, 2009, at 10:36 PM, Meredith L. Patterson wrote:
>
>
>
> > And it's been boingboinged.
> >http://www.boingboing.net/2009/02/06/crowdsourced-science.html
>
> > Go Tito!
>
> > --mlp
>

> > On Sat, Feb 7, 2009 at 3:58 AM, Tito Jankowski <titojankow...@gmail.com

[Meredith L. Patterson]

On Sat, Feb 7, 2009 at 12:20 PM, Steve Dickie <falcon...@gmail.com> wrote:
>
> I used to be a taxonomist (in a former life) now I'm a high school
> physics teacher. I have a couple quick questions. Hopefully they
> aren't too ignorant, I haven't done any DNA work in about ten years.
> First I want to say this is totally awesome and I will be passing this
> along to other teachers I know.
>
> Do all the straws run at the same rate? If not, how do I know which
> band I want?

I think the key there will be making sure that all the straws are
exactly the same length -- each straw behaves like a resistor, so just
like any other resistive material, a greater amount of material will
mean a higher resistance (and thus lower current at constant voltage).

> How do you stain for DNA in a straw? All the protocols I used (again,
> 10 years ago) involved staining the gel after a run.

Easiest way to do that would be to use a stain that you add to the
warm agarose before pouring, such as SYBR Safe or GR Safe (or ethidium
bromide, but the cool kids don't use that anymore).

I suppose you could slit the straw open with a razor blade if you
wanted to use methylene blue, but that sounds like a huge pain in the
ass.

--mlp

[Meredith L. Patterson]

On Sat, Feb 7, 2009 at 1:38 PM, Meredith L. Patterson
<clon...@gmail.com> wrote:
> On Sat, Feb 7, 2009 at 12:20 PM, Steve Dickie <falcon...@gmail.com> wrote:
>> Do all the straws run at the same rate? If not, how do I know which
>> band I want?
>
> I think the key there will be making sure that all the straws are
> exactly the same length -- each straw behaves like a resistor, so just
> like any other resistive material, a greater amount of material will
> mean a higher resistance (and thus lower current at constant voltage).

We could of course find out for sure by doing a bunch of test runs of
nothing but molecular weight markers.

--mlp

[Bryan Bishop]

On Sat, Feb 7, 2009 at 6:38 AM, Meredith L. Patterson wrote:

> On Sat, Feb 7, 2009 at 12:20 PM, Steve Dickie wrote:
>> How do you stain for DNA in a straw? All the protocols I used (again,
>> 10 years ago) involved staining the gel after a run.
>
> Easiest way to do that would be to use a stain that you add to the
> warm agarose before pouring, such as SYBR Safe or GR Safe (or ethidium
> bromide, but the cool kids don't use that anymore).
>
> I suppose you could slit the straw open with a razor blade if you
> wanted to use methylene blue, but that sounds like a huge pain in the
> ass.

There's also the option of working on the optics-based solutions :-).

http://groups.google.com/group/diybio/msg/52fe02cd7870cc2e

But at the moment, if you have stains laying around, it's easier to stain.

- Bryan
http://heybryan.org/
1 512 203 0507

[ben lipkowitz]

On Sat, 7 Feb 2009, Meredith L. Patterson wrote:
> I think the key there will be making sure that all the straws are
> exactly the same length -- each straw behaves like a resistor, so just
> like any other resistive material, a greater amount of material will
> mean a higher resistance (and thus lower current at constant voltage).
>
>> How do you stain for DNA in a straw? All the protocols I used (again,
>> 10 years ago) involved staining the gel after a run.
>
> Easiest way to do that would be to use a stain that you add to the
> warm agarose before pouring, such as SYBR Safe or GR Safe (or ethidium
> bromide, but the cool kids don't use that anymore).
>
> I suppose you could slit the straw open with a razor blade if you
> wanted to use methylene blue, but that sounds like a huge pain in the
> ass.

here's another idea:

Before filling the straw with gel, place a smaller straw inside and well
centered. Load the DNA into the ring-shaped cavity between the walls.
Then, when you want to stain, just pull the inner straw out and suck
methylene blue into the empty "core".

[Meredith L. Patterson]

On Wed, Feb 11, 2009 at 12:21 AM, ben lipkowitz <fe...@sdf.lonestar.org> wrote:
> here's another idea:
>
> Before filling the straw with gel, place a smaller straw inside and well
> centered. Load the DNA into the ring-shaped cavity between the walls.
> Then, when you want to stain, just pull the inner straw out and suck
> methylene blue into the empty "core".

Oooh, good one! Though safety concerns prompt me to issue the
reminder, never pipet by mouth. (There are squeezy-bulb things that
will do the job just fine.)

A bubble-tea straw and a regular straw would do the job, I think. Toric gels!

--mlp

[efer...@gmail.com]

Whoa! Straws in straws :) great idea
Sent from my Verizon Wireless BlackBerry

-----Original Message-----
From: ben lipkowitz <fe...@sdf.lonestar.org>

Date: Tue, 10 Feb 2009 23:21:55
To: <diy...@googlegroups.com>
Subject: Re: Keiki gels: Gel electrophoreis in a straw

[William Heath]

Hi All,

Not sure your aware but I made this work at noisebridge last night with food coloring!  I would like to now use real dna but I am not aware of how to get the dna etc...  Also how does the DNA show up in the gel exactly?  Do I just spit in the little hole that I put the food coloring in before? :)

-Tim

P.S.

DIY BIO ROCKS!  Tell me more things I can try!  Can I like take the dna and apply pcr to it?  How do I do that etc... ?

[Cory Tobin]

> Also how does the DNA show up in the gel exactly?

To make it show up you have to stain it with some chemical, then
usually illuminate it with a certain wavelength. The traditional way
is to stain w/ ethidium bromide, then view it with UV. Now days there
are other, more expensive, chemicals (SYBR Green, SYBR Gold, SYBR
Safe, etc). Here is a gel I ran today. The gel was pre-stained with
ethidium bromide, illuminated w/ UV-B...
http://www.its.caltech.edu/~ctobin/rtPCR_2009_2_10.png

> Do I just
> spit in the little hole that I put the food coloring in before? :)

You'd probably want to purify the DNA from the mucus and cells. A
while back I think someone posted a simple protocol for doing a crude
dna purification from spit. Maybe try that? Else, in anti-DIY
fashion, you could buy some. I usually use NEB's 1kb ladder (
http://www.neb.com/nebecomm/products/productN3232.asp ). That is what
I used in the picture I posted, far left lane.

> DIY BIO ROCKS! Tell me more things I can try! Can I like take the dna and
> apply pcr to it? How do I do that etc... ?

Sure. You have to purify the DNA from the gel first. I haven't seen
any DIY protocols for this yet. The most widely used one these days
is Qiagen's gel extraction kit (
http://www1.qiagen.com/Products/DnaCleanup/GelCleanup.aspx ). The DNA
eluted in this protocol can be used directly in PCR.

> Easiest way to do that would be to use a stain that you add to the
> warm agarose before pouring, such as SYBR Safe or GR Safe (or ethidium
> bromide, but the cool kids don't use that anymore).

I wouldn't recommend adding the SYBR dyes directly to the molten
agarose. From what I have tested, both SYBR Green and SYBR Gold slow
down the migration of the bands. It may be a linear effect, but I
haven't made any precise measurements. I just stick to poststaining
w/ the SYBRs. Oh, and by the way, the cool kids _do_ still use
ethidium ;-) Its cheap, bright, and there is no problem adding it to
the agarose prior to casting.

Also, I noticed the protocol used for this food-coloring separation
uses regular agar. If anyone has good results separating DNA with
agar, please tell me. Agar is so much cheaper than agarose.

Cory

[Rajagopal]

I hate the AD for Verizon Blackberry...It shows your interest in
communication is ok but your real work in DIYBIO is not evident ...
Pl remove ur tool"s aggrandisement
Raj

[Tom Knight]

DNA absorbs UV light, so it can be visualized by shadowing. Typically
this is done by illuminating a gel over a fluorescent plate (often a
thin layer chromatography plate) and observing the shadow. Presumably
a similar thing could be done with the straw. This is also the way
HPLC detectors observe the chemical peaks coming out of the column.

[Lauriel]

This straw electrophoresis seems like a neat idea, but would there be
a way to tell the size of the DNA? How would you run a marker?
-Lauriel

[William Heath]

Hi Anyone want to get together this Saturday to do more diy bio experiments at Noisebridge?

-Tim

[Tyler DeWitt]

Extracting the DNA from a few buccal (cheek) cells in your spit isn't
going to give you enough DNA to see on a gel. Plus, during these sort
of purifications the genomic DNA usually shears in multiple places, so
you wouldn't even be able to get a consistent banding pattern.

You've got a to have a lot of DNA to analyze it on a gel. That's why,
for example, if you were DNA fingerprinting someone, you'd PCR
sections of the genomic DNA to make zillions of copies of a specific
fragment before you ran it on a gel. I'm not sure whether DIY DNA
extraction techniques (usually with soap, salt, and liquor) produce
PCR-ready DNA. That's something I'll put on my list to try in the next
few days.

On Feb 10, 9:20 pm, William Heath <wghe...@gmail.com> wrote:
> Hi All,
>
> Not sure your aware but I made this work at noisebridge last night with food
> coloring!  I would like to now use real dna but I am not aware of how to get
> the dna etc...  Also how does the DNA show up in the gel exactly?  Do I just
> spit in the little hole that I put the food coloring in before? :)
>
> -Tim
>
> P.S.
>
> DIY BIO ROCKS!  Tell me more things I can try!  Can I like take the dna and
> apply pcr to it?  How do I do that etc... ?
>

[Tyler DeWitt]

I suppose there'd be a few possibilities. Taking into mind Meredith's
resistor comment from earlier, you could get two straws identical in
length, run a ladder in one and your sample in the other.

However, it would also be possible to run both your sample and the
ladder in one straw. Obviously, multiple fragments of different size
are run all the time in the same lane: that is what a marker is, after
all. However, it could be a little sloppy: you'd want to confirm that
your sample and and your marker both ran OK by themselves before you
mixed them together, because screwy results in a mixed lane would be
harder to troubleshoot.

[Dan]

You also have to make sure that the V/mm gradient is very consistent
across the box if you want to run straws in parallel. That's why gel
boxes use long wires as the electrodes to ensure that the DNA at
location A runs at close to the same speed as location B. I would
love to see some testing of multiple straws for this. My guess is
that there will be significant issues in getting DNA in more than one
straw to go at the same speed. It's a decent way to get rough size
data (which is often good enough, knowing something is ~7kb) but size
determination is something I'd be hesitant to do without a lot of
testing.

[Cory Tobin]

> However, it would also be possible to run both your sample and the
> ladder in one straw.

So if your ladder had a 2kb band and your sample had a 2kb band, how
could you tell that your sample had any DNA in it? The bands would be
in the sample place. Plus, if you wanted to purify your band, you
would end up with the ladder in there as well.

-Cory

[Tyler DeWitt]

Cory,

It's an admittedly very crude method. But if I just needed to check
the size of a fragment, though, the chance of obtaining completely co-
running bands is a risk I'd be willing to take.

Let's see . . . what would a more elegant solution be? How about
sticking a long flat piece of plastic down the middle of the straw
before you poured in the agar, creating essentially two separate
lanes? I doubt the potential difference between the two sides of the
straw would be enough to result in significantly different migration
speeds.


t

[sgt york]

DNA doesn't have to be super-clean for PCR. I used to have a technique
for single-fly PCR that was as follows:

Put fly in 1.5mL tube w/ 20ug/mL proteinase K dissolved in 100uL TE
(10mM Tris, pH 8 with 0.5mM EDTA; a standard DNA storage buffer)
Homogenize with a pestle.
Incubate overnight at 55C (DNAses don't work at 55C, but proteinase K
does).
Next day, boil 5 minutes to inactivate proteinase
Take 1uL for PCR.

Always worked for me, and I probably screened a hundred lines a week
this way for nearly a year. I've seen a similar protocol for
extraction from mouse toes (never used it, though), and I've used a
modified version of this technique to get DNA from single bacterial
colonies.

[ben lipkowitz]

this is stupid. just run the straws in the same chamber with the same
electrodes from the same batch of agarose at different times. think
"gel spectrometer calibration" not "horse race"

[Josh Perfetto]

This might be ok but only under some use cases and only if you can deal with
a very rough interpretation of the fragment size. One problem is that even
if the bands are not coincident, they may be very close, and you won't know
which band is the ladder and which is the sample, giving you only a rough
indication of fragment size and making this unsuitable for cutting out
bands. You may also be running samples where there are multiple fragments,
or some of the DNA was supercoiled, or for whatever reason you get multiple
bands where there was supposed to be a single band, making it even harder to
determine which band is which. There may be circumstances where this is
acceptable, but since electrophoresis is frequently used either to cut out
bands, or to verify that something has worked before going on to further
manipulations, I don't think this is very good as a general solution.

I am not sure, but I think the difference in diameters of the two straws,
particularly if one was much smaller than the other, may impact the rate at
which DNA runs in the tubes and may cause more blurring in the smaller tube.
Having one straw within another would also impact the visibility of bands in
the inner tube somewhat as well, since the straw won't be entirely
transparent.

I think you should be able to get multiple tubes to run at the same rate, so
long as you create a uniform electric field within the chamber. This can be
done by ensuring your electrodes are parallel to each other and
perpendicular to the tubes, and that everything else is uniform (i.e.
consistent dimensions of the electrodes, tube diameter, etc.) You also
probably want to make sure the electrodes extend a tad beyond the outermost
tube.

There would probably still be some risk that one tube was misaligned
slightly, but this may be a trade-off you can make if you find tubes to be
easier to work with than full gels. To be quite honest I am still not
seeing the advantage of tubes over gels if you are doing horizontal gels, is
it just that you can use the tubes as a casting apparatus? It just seems to
me that casting a horizontal gel is so easy, while pouring agarose down a
straw while preventing an air bubble from forming would be painful, but
maybe I am missing something.

-Josh

[Cory Tobin]

Just curious, what is the advantage of using straws over a rectangular slab?

-Cory

[Lauriel]

I don't mean to be a downer, I love creativity and innovation, but I
don't see why straws are better then a gel rig? Gel electrophoresis
already is fast and convenient, so what does this have going for it
that is better then what is already available?
-Lauriel

[ben lipkowitz]

When I was using gels we had to pick it up by hand and slosh it around in
a tank of EtBr, then flop it onto a transilluminator and take a picture.
This was a messy process and you had to be careful not to break the gel.
You have to heat up your beaker of agarose each time, inevitably changing
the concentration, then there's big tanks of buffer to mess around with.
(Can anyone explain why we need big tanks of buffer?)

You can't store gels in the fridge because their large exposed surface
area will dry them out, and handling is an issue since they're fragile.
Make sure you don't have a bubble sticking to the gel comb! and definitely
don't spill even the tiniest bit of DNA outside the well if you're going
to try to amplify any of the fragments! (easier said than done, while
looking at something a couple mm across, half-transparent and underwater.)
And don't try to move the gel tank or it might just slosh around and
cross-contaminate the wells on its own.

Wouldn't it be nice if instead we had a piece of cheap equipment that was
repeatable, convenient, mess- and worry-free? A DNA spectrometer in every
lab!

The thing about a straw is that it's rigid and easily handled. This means
you can put it into a jig that holds the straw in the same place each run.
All the straws are identical in shape so they should experience identical
conditions during the run, and this means you can calibrate your machine
in terms of something easily measured, like time or integral of current,
instead of its position relative to some other bands on the gel.

Perhaps you think that fancy image processing software is the way to do
it. Several years ago when I used automatic band-detection software I was
not impressed. It was just as easy and more accurate to measure it by eye.

I'm disappointed by the lack of automation in typical biology experiments.
I was trying to find a way to automate the process of looking at a gel and
determining the "spectrum" or size distribution of DNA in the lane,
preferably without having to use any expensive or toxic (hard to acquire
and dispose) stains. The idea was to use a UV LED and a sensor to "see"
the DNA as it goes by. Somehow the concept of a built-in spectrophotometer
got lost in the excitement. Won't someone at least comment on this idea?

-fenn

[Nick Taylor]

Putting gunpowder and shot into a cartridge made machine-guns possible... I imagine there may be similar sorts of advantages to having the gel (which was formerly... well, gelly) turned into an easily swappable, transportable standardised part. The fact that this is a low-tech solution kindof makes it a double win.

I would've thought.

[Tom]

The straw trick is cute, but the problem with running DNA may turn out
to be resolution, you are probably not going to get much in the low kb
range that with agar and will need some more purified form of agarose.
Who knows what kind of DNAse or other enzymatic contamination may be
in that agar that will just eat your sample alive. One can pre-cast
and store loads of agarose gels in a tupperware container of running
buffer in a fridge for a long time. This is a wheel that has already
been invented. The cheap cost ($0) of doing it this way will be offset
by the cost in time it takes to perfect it. Purity of reagants and
sterile technique are going to be the basis of any molecular genetics
protocol.

[JonathanCline]

On Feb 12, 1:28 am, Lauriel <komo...@gmail.com> wrote:
> I don't mean to be a downer, I love creativity and innovation, but I
> don't see why straws are better then a gel rig?  Gel electrophoresis
> already is fast and convenient, so what does this have going for it
> that is better then what is already available?
> -Lauriel

Presumably, the gels with lower cross sectional area will need less
current for running the gel, which could allow for (1) much easier
power supply design at high voltages, since total power is much
lower; (2) reduction of temperature, since total power is low; the
high voltage+current cause heat and breakdown of the agarose, which
prevents super-fast runs; (3) alternative protocols & reagents like
faster-better-media which allow very fast run times at very high
voltages; (4) maybe a cheap-tool method of extracting DNA from a line
(snip the straw) if this needs to be done; (5) ?others?

High voltages == ~1kV or greater. All of this needs experimental
proof of course. Cooling could be much easier. The gel box 2.0
design includes some plans for peltier device for cooling the flat
gel. Perhaps the proper term for this technique is millifluidics.
Not quite microfluidics, however smaller scale may give good
benefits. If any of the above is incorrect, then please correct me.

The comparison is minigels? Which are flat, though also pre-packaged,
similar to the idea of prepacked straws. Also seemingly more
expensive than they should be..

## Jonathan Cline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################

[sjst...@gmail.com]

> Presumably, the gels with lower cross sectional area will need less
> current for running the gel, which could allow for (1) much easier
> power supply design at high voltages, since total power is much
> lower;  (2) reduction of temperature, since total power is low; the
> high voltage+current cause heat and breakdown of the agarose, which
> prevents super-fast runs; (3) alternative protocols & reagents like
> faster-better-media which allow very fast run times at very high
> voltages; (4) maybe a cheap-tool method of extracting DNA from a line
> (snip the straw) if this needs to be done;  (5) ?others?
>

> ## Jonathan Cline


Sounds like you just described the Hundred thousand dollar
capillary electrophoresis machine in the last lab where I worked.
That's
essentially where this would be going, right? A DIY version of those
machines?