Paper electrophoresis... super available molecule separation?

[Nathan McCorkle]

So my thinking is everyone can get paper of some form, but not
agar/agarose... seems it's not ideal for DNA but it is for amino acids
and small proteins... it might be useful for DNA or large proteins in
certain assays though... i.e. yes/no assays or something

here are some things i dug up:

mentions a few paragraphs of history
https://www.idtdna.com/pages/docs/educational-resources/gel-electrophoresis.pdf

including
"
While paper and other solid support materials proved to be an
advantage over free
solutions for the electrophoretic analysis of biomolecules, gels were
adopted later
because gels not only minimized diffusion better than paper supports
they actually
participated in the separation process by interacting with the
migrating particles.
"

and referencing this article about Bothrops (pit viper snake) venom,
but it's in German (I think) and I can't read it (google translate
doesn't do much either because copy-paste don't keep the sepcial
characters)
http://nathanmccorkle.com/pdf/this%20might%20be%20about%20paper%20electrophoresis.pdf

I got thinking because I saw this chromatography paper for $0.65 per
foot and mentions "widely used for electrophoresis"
http://store01.prostores.com/servlet/thescienceshop/the-2012/CHROMATOGRAPHY-PAPER-2cm-Wide/Detail


thesis on
"Development of Paper electrophoresis technique for observation of
microgram quantities of protein" and "electrophoretic and
ultracentrifugal studies of soluble antigen-antibody complexes as a
method of determining antibody and antigen valences"
James T. Bradbury, California Institute of Technology, 1956
http://thesis.library.caltech.edu/995/1/Bradbury_jt_1956.pdf


--
-Nathan

[Cathal Garvey]

Hm, with the right buffering it might be possible to get this to work
with DNA, too. You want a buffer that interferes with the pi-bonding
that attracts DNA to cellulose fibers.

With chemical interactions out of the way, high-grade cellulose looks a
lot like agarose, chemically speaking. In fact, running a "gel" through
a lump of microbial cellulose has been a low-grade item on my to-do list
for a long time. Biggest barrier right now: I killed my kombucha
(G.xylinus) cultures long ago. :P

Would love to know if this would work.

[Koeng]

That would be wonderful if it was true!!! Someone should test this...

[Jeswin]

On Sat, Jan 19, 2013 at 7:37 PM, Cathal Garvey
<cathal...@cathalgarvey.me> wrote:
> Hm, with the right buffering it might be possible to get this to work
> with DNA, too. You want a buffer that interferes with the pi-bonding
> that attracts DNA to cellulose fibers.
>

For diybio purposes, it might be a good trade-off between cost and
effectiveness if it can work.

I think back in the day, they found that gels were probably faster
and gave better resolution. Since money was not much concern for
scientists (lot of grant money), everyone adopted gels. Jut my theory.

[Lawrence]

Here are 2 articles and chapters from the book "The Amateur Scientist" by C.L.Stong 1960". They can be found here:

http://jesseenterprises.net/amsci/1955/08/1955-08-fs.html - deals with paper electrophoresis

http://jesseenterprises.net/amsci/1953/02/1953-02-fs.html - deals with paper and column chromatography

They are old articles but are very informative. They show how to setup the systems and what to use. Sometimes the old ways can work for us just like they did for the scientists from a time before all the new techniques were created.

Lawrence
citsci.blogspot.ca
Citizen of Science

[shreyans chordia]

the idea sounds cool.. it would be really cheap if we could make bacteria produce cellulose fibres and then use them to run samples.. 

Although a drawback of that would be that the secreted proteins in the cellulose fibres will contaminate the sample we want to run..  Anyway to overcome that?   

[Cathal Garvey]

Proteases, etc.

Bacteria already make high-quality cellulose, at a higher purity than
plants do, in fact. It has a slimy consistency but it is exceptionally
strong.

The species most often credited with making "nature's best cellulose" is
Gluconacetobacter xylinus, shortened to G.xylinus, often called
"A.xylinus" in the literature for historical reasons (used to be just
"Acetobacter"). You can get the best strains for cellulose production by
buying a kombucha SCOBY on ebay and isolating for acid-producing
colonies on agar.

There have been some experiments getting E.coli to express the cellulose
operon from G.xylinus, and I seem to recall it worked out OK. So there's
definitely scope for producing it in another host, although G.xylinus is
already very easy to grow and efficient at producing cellulose, so
there's not that much need.

> <http://thesis.library.caltech.edu/995/1/Bradbury_jt_1956.pdf>
>
>
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[Nathan McCorkle]

On Jan 28, 2013 5:12 AM, "shreyans chordia" <shreyans...@gmail.com> wrote:
>
> the idea sounds cool.. it would be really cheap if we could make bacteria produce cellulose fibres and then use them to run samples.. 
> Although a drawback of that would be that the secreted proteins in the cellulose fibres will contaminate the sample we want to run..  Anyway to overcome that?

Pre-run the electrophoresis maybe

[shreyans chordia]

Interesting..  thats like running ampholytes before loading samples on a pH gradient gel..  only disadv is buffer wastage.. 

using proteases is not cost effective, also the proteases cannot get to the proteins because of the crystalline packing of cellulose.. 

how bout exploting the fact that cellulose can take high temps.. then we could ppt proteins out o it maybe.. but it could also clog the pores in the fiber..

gonna lookup bac cellulose processing, although i have a feeling most o it must be under wraps due to patenting :(

[Dakota Hamill]

Has anyone loaded up some paper with DNA, like a TLC plate, and let it run?  Next gel I run I'll give it a shot..though some thought should probably be put into how the electrodes get hooked up, so I don't have a soaking wet piece of paper with .5A running through it at 100V.  Yay science

Maybe a longer run at lower voltage would be better

[Sebastian Cocioba]

Ill run one when I get home too. How should we dispense the dna onto the paper? Do you think printer paper left to soak for a while would work? Tissue paper? Whatman? I fear the sheer density of any papet would result in low transit.

On Tuesday, January 29, 2013, Dakota Hamill wrote:

Has anyone loaded up some paper with DNA, like a TLC plate, and let it run?  Next gel I run I'll give it a shot..though some thought should probably be put into how the electrodes get hooked up, so I don't have a soaking wet piece of paper with .5A running through it at 100V.  Yay science

Maybe a longer run at lower voltage would be better

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[Dakota Hamill]

I was thinking sort of like TLC

http://www.dnatube.com/video/1845/Thin-Layer-Chromatography-TLC

But...with a voltage applied.  I think DNA would get stuck to silica. Can't think right now, gatta go

[Nathan McCorkle]

On Tue, Jan 29, 2013 at 2:03 PM, Sebastian Cocioba <scoc...@gmail.com> wrote:
> Ill run one when I get home too. How should we dispense the dna onto the
> paper? Do you think printer paper left to soak for a while would work?
> Tissue paper? Whatman? I fear the sheer density of any papet would result in
> low transit.

Maybe spot the DNA TLC-style on dry paper, sandwich that between two
microscope slides, seal the long edges with wax immerse... then put
the glass sandwich between two buffer tanks containing the electrodes.
You'd probably need to make gaskets for either end of the glass
sandwich though.

Any other ideas? I feel like you need glass plates because I think it
would restrict the DNA from diffusing into solution.

--
-Nathan

[Sebastian S. Cocioba]

I see where you are going with the slide sandwich idea. I have some spare ladder and will run it like you said in my gel box. Parafilm along the edges might work, no? Im really curious to see how my insta-stain etbr stickers will work on the paper...I'll have my results in 3hrs. Ill run the paper at low voltage since my box is tiny (gibco horizon 48 @ 25v at IDK milliamps).

Sebastian S Cocioba
CEO & Founder
New York Botanics, LLC

Sent via Mobile E-Mail

[Nathan McCorkle]

On Tue, Jan 29, 2013 at 3:45 PM, Sebastian S. Cocioba
<scoc...@gmail.com> wrote:
> I see where you are going with the slide sandwich idea. I have some spare ladder and will run it like you said in my gel box. Parafilm along the edges might work, no? Im really curious to see how my insta-stain etbr stickers will work on the paper...I'll have my results in 3hrs. Ill run the paper at low voltage since my box is tiny (gibco horizon 48 @ 25v at IDK milliamps).
>

hmm, maybe the sealing the sides isn't so much of a concern, but I'd
definitely still do the glass sandwich. I've only ever had parafilm
react to organic vapors, I imagine it would hold up pretty well in the
electrphoresis chamber

--
-Nathan

[Dakota Hamill]

I did a quick run with marker dyes 10 minutes ago as I didn't have any DNA or food coloring.  Tried it once with 1x TBE and once with salt water.  Once without the glass sandwich and once without the glass sandwich.  The first picture was just to make sure the dyes dispersed in all directions without a voltage applied.  

 

When I ran them I completed the battery circuit, sometimes using the automotive bulb as a resistor to keep current @ no more than 400mA.  I tried using my gel box supply but it was giving leak load errors and other BS so I didn't even bother.  

Basically...the results were completely useless since I never had a good control, and the one time it looked like the dyes were migrating, the slide was slanted and gravity had its way with them.  I was also impatient and didn't wait very long.

BUT.  I can say the glass slide sandwich is much nicer to deal with, and capillary action inside it allows for the electrolyte to quickly meet in the middle.  I dotted in the middle and then dropped electrolyte at the lip on both the + and - ends and they met in the middle.  

With DNA, you could assume it'd be moving away from - towards positive, but I didn't know the charges of the dyes off hand so starting in the middle seemed best.

http://imgur.com/a/dLjf5#4

Anyway, pretty useless but it looks pretty.  I'll be interested to see how Sebastian's run comes out.   I'll try again later if I can get the gel box to work.  I assume 9V was not enough to make them run fast.

GO TEAM

[Sebastian S. Cocioba]

So I cut out a slide sized piece of coffee filter paper (sadly unbleached and lightly brown) wrapped it with two strips of parafilm but made sure the narrower edges were exposed. I dispensed 10uL at the negative pole edge near the center of the opening. It dispersed about a half inch down the paper slide sandwich. I made two samples and put them in my tiny electrophoresis chamber at 25v. A normal 1% gel takes 3 hours at this setting so im gonna let it run for that long. Ill check every hour and will post on imagur once its done.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

--

[Koeng]

I tried this myself as well, because I have never preformed electrophoresis (agarose in the mail!), I thought it might be easier for my to pick up. I will post pictures when I am done.

Basically I put some loading die on a straight line against some normal printer paper. A microscope slide blocked it from going through the rest of the paper. Then I dipped it with only the bottom slide on into some TBE buffer. Once it was soaked, I put the top on,  then I left it inside the electrophoresis apparatus at 50v (Since I want to go to sleep soon)I will check it every thirty minutes.

-Koeng

On Saturday, January 19, 2013 4:11:43 PM UTC-8, Nathan McCorkle wrote:

[Sebastian Cocioba]

The gel run for the first sample was inconclusive. The tracker did not move beyond the initial dispersion. I stained the strip with an EtBr Insta-Stain pad. No sign of bands...or anything for that matter. The strip did glow a bit orange so maybe the filter absorbed all the etbr? I let the other strip slide sandwich keep running and cranked the voltage to 100v. Ill check that one for movement in one hour. 

Here is a pic of the strip prior to stain after the initial run. Note the smear of tracking dye on the right. It did not move much if at all. Strip post run

I'm gonna get white filter paper tomorrow and play with permanent marker dots in the chamber to see if there is any sign of motion. I think the surface area of the strip touching the buffer is low thus lower current through the paper. Could this be the issue? If so, a much higher voltage may be needed. More testing to come...this was a fun example of remote experimentation. Go team, indeed. :)

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[Patrik D'haeseleer]

Hey Cathal - do you have some Kombucha going right now? I'd love to see what happens if you take a nice thick "mother" SCOBY, cut some wells into it, and use it as an electrophoresis gel. :-). Food dyes electrophorese pretty well.

Patrik

[Sebastian S. Cocioba]

So I tried it again with loading dye without wrapping it in parafilm and it worked. The loading dye moved across the strip without smearing but the transit distance was no where near where it would have been had the medium been agarose. 

I put 5uL of concentrated loading dye near the end of the strip of coffee filter paper. I set the strip between two microscope slides and carefully laid the slide sandwich onto the chamber floor and filled it with standard TAE buffer. I ran the sandwich at 25v for one hour just to see if the dye would budge. 

The main issue I see is the lack of dispersion control in the initial loading and the staining of the strip post run with EtBr. Im going to get some white filter paper and try to stain the dna ladder post run once more. It would be very interesting if this works.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

On Jan 30, 2013, at 1:06 AM, Patrik D'haeseleer <pat...@gmail.com> wrote:

Hey Cathal - do you have some Kombucha going right now? I'd love to see what happens if you take a nice thick "mother" SCOBY, cut some wells into it, and use it as an electrophoresis gel. :-). Food dyes electrophorese pretty well.

Patrik

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[Cathal Garvey]

Perhaps you could use "graphite electrodes" by drawing them onto either
end of the paper with a B6 pencil? :)

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[Dakota Hamill]

Very cool Sebastian,  are you using methylene blue loading dye?  I think that runs proportional to 1kb DNA strands, at least in agarose.  I never even thought to actually just lay the slide sandwich down into the gel box and run it that way, now I can throw out the alligator clips, if I can get this damn gel box to stop leaking. 

What about taking a long rectangular strip of the filter paper, melting some candle wax in a longish tray, then dipping the long ends of the filter paper in a little on both sides, so you have this strip running right down the middle that is un-waxed and theoretically, just a skinnier rectangle, bordered by wax.

Trying to think of other ways to seal off a narrow channel, to control the initial dispersion as you said, but keep open a running lane for the DNA or dye to migrate.

[Cathal Garvey]

Sadly I let my Kombucha stale in the fridge for too long and couldn't
recover it. And, my isolated culture of G.xylinus is long dried out too,
due to a gap in the parafilm. So, I'll have to re-order Kombucha and
re-isolate G.xylinus! Thankfully, the latter isn't too challenging.

You make agar containing calcium carbonate powder, stir and pour right
before solidifying so you get evenly dispersed CaCO4 particles through
the gel, then streak and re-streak your kombucha on the surface. The
colonies that create a broad area of clear agar (acid dissolving the
CaC04) are your Geo/Acetobacter, of which there may be one or two species.

Isolate those and grow to stationary, the plates that form nodules of
whitish cellulose after a week or so are your guys. :)

I have the agar recipe lying around somewhere, will dig up soon.

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[Koeng]

My results with the printer paper-

The dyes spread out and the paper looked kinda like a watercolor. No lines whatsoever. Printer paper doesn't work. That is what I concluded... maybe you guys can try it

On Saturday, January 19, 2013 4:11:43 PM UTC-8, Nathan McCorkle wrote:

[Brian Degger]

Some paper is coated, I tried cartridge art paper and that too wasn't good.

Anyone tried coffee filter paper?

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--
----------------------------------------
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http://makerspace.org.uk

http://transitlab.org

----------------------------------------

[Sebastian Cocioba]

See my previous post. It worked.

[John Griessen]

On 01/30/2013 07:48 AM, Dakota Hamill wrote:
> Trying to think of other ways to seal off a narrow channel, to control the initial dispersion as you said, but keep open a running
> lane for the DNA or dye to migrate.

It seems to me that ordinary filter papers wick rapidly, making wide blurs out of any sample put on them.

If you coated filter paper with agarose then dried it be convenient paper again, it might
perform more like agarose -- low dispersion. If drying somehow ruins it,then store
it wet refrigerated/frozen and you'd get the agarose narrow dispersion
benefits with a small amount and the convenience of paper.

It might pay to try different papers. Clay coated papers for photo printing might be good.
Reading about TLC made me try searching for silica gel paper and it exists:
http://www.fishersci.com/ecomm/servlet/fsproductdetail_10652_785049__-1_0
and is thick, which is good for electrophoresis.

The electrophoresis voltage will have to be higher than with gel box agarose
since it is such a "paper thin" path, therefore less conductive for the same conditions.

Here is something from TLC:
http://85.238.144.18/lifescience/literature/061009_Making_TLC_Plates_from_Bulk_TLC_Silica_Gels.pdf

"The fine silica will not stay on the plate after coating if some type of binder is not
included. . . . The classical binder used is gypsum . . . In most silica gels it is put in
at a level of from 10-15% to give good binding to the glass plates . . . For a
stronger layer, polyvinyl alcohol or polyvinyl pyrollidone can be added to a TLC grade silica gel
(without other binders) in a level of 1-2% by weight as a polymeric binder . . . Heat drying after
air-drying makes cross-linking of this type of polymer binders.

What if you take fine filter paper strips, make a slurry as for TLC, pull paper through slurry
of silica gel and gypsum plaster, then a double squeegie, (like used to dry photo film),
to get a uniform amount on the paper?

Just some ideas. I can't stop to try these out myself just now.

[Jeswin]

On Wed, Jan 30, 2013 at 8:48 AM, Dakota Hamill <dko...@gmail.com> wrote:
>
> Trying to think of other ways to seal off a narrow channel, to control the
> initial dispersion as you said, but keep open a running lane for the DNA or
> dye to migrate.
>

I don't think we should think of paper electrophoresis in relation to
gel electrophoresis. For example, in gel electrophoresis, we run
multiple lanes at once. For paper, maybe it's best to run 1 sample per
strip. That will prevent the diffused molecules from merging together.
Multiple strips of the same length can probably be run together and
still be good for comparision. The voltage through all the strips
should be the same and so should the rate of migration. Correct me on
this last statement.

[Nathan McCorkle]

I think the distance between the electrodes makes the most impact


--
-Nathan

[Dakota Hamill]

Yeah I only meant 1 sample per strip.  Think of a car hood with a racing stripe down the middle.  The racing stripe is the lane / path the DNA will be loaded onto one side of, and the rest of the paint of the hood (the rest of the paper on either side of the lane), is something that seals the paper and prevents diffusion of the sample, be it wax, or as john mentioned, dried agarose. 

I'll give it a shot later with dyes and some candle wax sealing a lane, and if that doesn't work maybe use some agarose.  Wish I had a way to visualize my DNA on hand but, no go right now.

I'll see if I have any stainless steel wire around to try and rig up mobile electrodes to vary the distance.

[Nathan McCorkle]

Congrats on trying this and it working!

Here are some papers I just dug on regarding nucleic acids (mono and
poly-mers), there's a lot of data on pH charge-dependence effecting
migration (but basically you want the pH above 3 or 4), also saying it
took an hour for Uridine 3'-phosphate (charge of -1 between pH 2 and
5) to move 8cm at 20V/cm:
http://nathanmccorkle.com/pdf/paper_electrophoresis/

including:
DETERMINATION OF ADENINE NUCLEOTIDES BY PAPER ELECTROPHORESIS.pdf
Paper Electrophoresis of Nucleic Acid Components.pdf
Practical Applications of Paper Electrophoresis.pdf
Techniques for elution of nucleic acid components from paper.pdf
The Use and Limitations of Filter-paper Electrophoresis.pdf

--
-Nathan

[Cathal Garvey]

The "mesh strength" of your gel/paper is also relevant, so if you're
using dense paper you would expect slower migration. Given that paper
varies very widely in quality, density and chemical purity, you'd
probably have to work the protocol to the brand of paper you're using
and try to stick to the same brand.

I wonder how big a difference it would make to use archival-grade paper,
which tends to have less bleaching byproducts/acids, less tannin, and
often higher-quality cellulose ~= longer cellulose fibers. Probably
you'd get a more even matrix of cellulose leading to more predictable
and even migration of bands, if it worked at all?

[John Griessen]

On 01/30/2013 11:45 AM, Nathan McCorkle wrote:
> I think the distance between the electrodes makes the most impact

Current through a sheet of uniform substance reduces to being about ohms per square.
A square of any size is still a square. A long, (in squares), or narrow, (in squares),
path is less conductive no matter what size it is.

[Nathan McCorkle]

But DNA migration depends on the e-field not on the power through it

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[John Griessen]

On 01/30/2013 05:49 PM, Nathan McCorkle wrote:
> But DNA migration depends on the e-field not on the power through it

Sure, that's true. And I was talking about the e-field also.
Ohms per square are the Ohms that resist the e-field. Look it up.

The aspect ratio of an electrophoresis path determines how conductive it is
if all other variables are the same, (salts, buffers, conductive stuff in solution,
gooeyness, amount of duct tape used, viscosity, Coriolis effect, what have you)

In short, neglect the rest, take the ohms per square analysis to get to the
heart of designing any new shape relative to a usual gel box.

How you reduce it to a 2D problem from a 3D world is to find the conductivity of a cube,
then map that onto your path layout in 2D and consider it a uniform layer that can be treated
as X,Y movement only, neglecting Z axis, and you're to a 2D representation that can
simplify and give you design insights.

[Nathan McCorkle]

On Wed, Jan 30, 2013 at 6:55 PM, John Griessen <jo...@industromatic.com> wrote:
> On 01/30/2013 05:49 PM, Nathan McCorkle wrote:
>>
>> But DNA migration depends on the e-field not on the power through it
>
>
> Sure, that's true. And I was talking about the e-field also.
> Ohms per square are the Ohms that resist the e-field. Look it up.

I'm not sure why you're mentioning current or the resistance of the
media, or what you want me to look up.

You only want to keep current low enough such that the media doesn't
become too hot, and that depends on ion (salt and buffer)
concentrations.

>
> The aspect ratio of an electrophoresis path determines how conductive it is
> if all other variables are the same, (salts, buffers, conductive stuff in
> solution,
> gooeyness, amount of duct tape used, viscosity, Coriolis effect, what have
> you)

Sure that's going to reduce the overall current, that's a good thing.
Automated Sanger sequencing systems use 50 micron capillaries to do
electrophoresis.

310 Capillary, 61cm x 50µm (50 cm well-to-read) - for sequencing
applications. Internally uncoated. 2 capillaries/package (100
runs/capillary).
https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catProductDetail&productID=402840&catID=602042&backButton=true

> In short, neglect the rest, take the ohms per square analysis to get to the
> heart of designing any new shape relative to a usual gel box.
>
> How you reduce it to a 2D problem from a 3D world is to find the
> conductivity of a cube,
> then map that onto your path layout in 2D and consider it a uniform layer
> that can be treated
> as X,Y movement only, neglecting Z axis, and you're to a 2D representation
> that can
> simplify and give you design insights.
>

You'd probably get close just using the analytical concentration of
the buffer, then stick an ohm meter on the electrodes.

But if you have a voltage controlled power supply, then you just worry
about Volts per distance?

Are we on the same page?


--
-Nathan

[Patrik D'haeseleer]

If you can get your hands on some, try the filter paper used for Northern/Southern/Western blotting. Very fine grain, very uniform, and no contaminants.

[John Griessen]

On 01/30/2013 09:44 PM, Nathan McCorkle wrote:
> On Wed, Jan 30, 2013 at 6:55 PM, John Griessen <jo...@industromatic.com> wrote:
>> On 01/30/2013 05:49 PM, Nathan McCorkle wrote:
>>>
>>> But DNA migration depends on the e-field not on the power through it
>>
>>
>> Sure, that's true. And I was talking about the e-field also.
>> Ohms per square are the Ohms that resist the e-field. Look it up.
>
> I'm not sure why you're mentioning current or the resistance of the
> media, or what you want me to look up.

I am offering to show you how to simplify thinking about design choices
for electrophoresis. What you can neglect when thinking about path length.

> You only want to keep current low enough such that the media doesn't
> become too hot, and that depends on ion (salt and buffer)
> concentrations.

The above are side issues of path length that you will "get" when you get that
the "length" does not affect resistance of a path of gel or whatever independently,
you need to consider the Ohms per square shape based resistance of a path to get
an independent variable you can use to get what you want, whether that be a
particular current, or power level, etc.

>> The aspect ratio of an electrophoresis path determines how conductive it is
>> if all other variables are the same, (salts, buffers, conductive stuff in
>> solution,
>> gooeyness, amount of duct tape used, viscosity, Coriolis effect, what have
>> you)
>
> Sure that's going to reduce the overall current, that's a good thing.
> Automated Sanger sequencing systems use 50 micron capillaries to do
> electrophoresis.
>
> 310 Capillary, 61cm x 50µm (50 cm well-to-read) - for sequencing
> applications. Internally uncoated. 2 capillaries/package (100
> runs/capillary).
> https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catProductDetail&productID=402840&catID=602042&backButton=true

All that above was on a side issue again, so back to defining the relative path shape
based effect on "a normal electrophoresis system":

>> simplify and give you design insights.
>>
>

> You'd probably get close just. . .just worry

> about Volts per distance?
>
> Are we on the same page?

Not yet. Sounds like you aren't patient enough, but here's another try:

With all other variables held constant, (which means stop talking about particular
designs of machines in specific and extract what is their path shape for purposes of
this discussion), the depth and length and width of a straight path for electrophoresis
analytes to travel on is going to make a big change on how fast they move at the same voltage applied.
In the case of paper The thinness of paper is a linear factor of change you can figure out quickly
if you consider a square area to get a measurement of ohms or current from when same volts per square
is applied. Volts per square goes back to the concept of conductivity of a square piece of a sheet
of known sheet resistance, (sheet resistance is specified in ohms per square).

Converting a particular Ohms measurement to "per square" is the first step when changing
things around a lot as in comparing a gel box to paper strip electrophoresis. Otherwise
you would get lost in details and find they are all interdependent.

A quick example is a gel box twice as long as wide. It has 2 squares of uniform
sheet area. On is the same as the other. Analytes moving along in one square move the same
as in the other. So the Ohms measurement
of volts end to end divided by current end to end is the end to end Ohms. The end to end Ohms
divided by 2, ( 2 squares), gives its sheet resistivity.

Sheet resistivity is useful to use in designing a new shape electrophoresis path for whatever
current, and therefore voltages you desire.

That's the page I've been on. Only a general concept page, no specifics.

Back to work.

[Dakota Hamill]

Well I'm confused now but.

Let's say we have a piece of filter paper 10cm long, 1 cm wide, and 0.1 cm thick. 

Is it wrong to think it could handle similar voltages and currents that an agarose gel could?

Say it was running at 100V and 0.050A.  Could you then use V=IR to calculate the resistance of the buffer soaked paper would be 2000 Ohms?

Or, via P=IV, P = (0.050A) x (100V) = 5W

And 5W = 5 J/s * 600 seconds (10 minute run) = 3000 Joules

That's a lot of energy to put through a buffer soaked piece of paper soaked between two glass slides, no?  Though I guess if it's submerged in the giant buffer tank it has a heat sink sort of.

I havn't done good ol' electricity basics in a while so I'm probably wrong in the 2000 Ohm part if not everywhere.

What I'm wondering is...WITHOUT trial and error, could you calculate the "perfect" conditions to run an agarose gel under - meaning voltage + current assuming a tris buffer?

Likewise, could we calculate the optimal conditions to run an electrophoresis 10,1,.1 cm thick piece of paper with tris buffer? 

[John Griessen]

On 01/31/2013 09:46 AM, Dakota Hamill wrote:
> What I'm wondering is...WITHOUT trial and error, could you calculate the "perfect" conditions to run an agarose gel under -
> meaning voltage + current assuming a tris buffer?
>
> Likewise, could we calculate the optimal conditions to run an electrophoresis 10,1,.1 cm thick piece of paper with tris buffer?

Probably this should go off list, then come back with results. Short answer, "Sure."

Dakota, how about you reply to me about some actual ohms measurements and I'll help you calculate
and compare. I'm in the middle of a non-bio project and won't make any agarose gel to measure it myself
anytime soon.

John Griessen

[John Griessen]

On 01/31/2013 09:46 AM, Dakota Hamill wrote:

> Let's say we have a piece of filter paper 10cm long, 1 cm wide, and 0.1 cm thick.
>
> Is it wrong to think it could handle similar voltages and currents that an agarose gel could?

No, that sounds plausible/possible.

> Say it was running at 100V and 0.050A. Could you then use V=IR to calculate the resistance of the buffer soaked paper would be
> 2000 Ohms?
>
> Or, via P=IV, P = (0.050A) x (100V) = 5W

Yep. That's plausible/possible. A 5Watt rated resistor is only 1 com long and .4 cm diameter
and it can get warm to hot at 5W, but not char to black. A 10 cm long strip 1cm wide might
be barely perceptibly warm in still air while dissipating 5W since its surface areas are so
much greater. Your guess at resistance could be way off, since gel boxes aren't so skinny
as you described. What if The 5 Watt level happened at a current of .005Amp? then the
Volts would need to be 1KV. I think that might be more normal for a 10X long as it is wide
gel or paper/electrolyte.

[Dakota Hamill]

http://imgur.com/a/t1SEv

Re-doing the experiment but with coffee filter paper, and a real gel box, and a real power supply.  As you can tell from the first picture, the chem filter paper I have allows the dye to disperse way too fast, as it should, since it's supposed to let solvents pass through it quickly.  Sebastian seemed to have decent sounding results using the brown coffee filter paper, so we'll see how it works with the dyes.

I tried to reduce pouring turbulence too much when pouring in the buffer but, was unavoidable when the solvent front rushed through the slide sandwhiches.  Running at 120V 100mA or there abouts.

There is already noticeable migration of one of the components of the markers to one direction---anyone fancy a guess?  You'll win the Marker Migration badge of honor.

I'll try to post a video later if you want to experience the most intense marker dye coffee filter electrophoresis you've ever seen.  Blow your mind if it was in 3D.

GO TEAM

Oh and the candles weren't to set the mood, was going to try that dipping strategy to section off a running lane and prevent bleeding.  Maybe next time

[Dakota Hamill]

Well I cut 3 strips of filter paper and put them on glass slides.  Soaked one in salt water, one in 1x TAE, one in 10x TAE.

All read way too high on the multimeter for resistance until I got to the 200k and 2 million Ohm setting.

I tried to keep the probes 1cm apart but came to realize it didn't really matter.  In general I could get a resistance reading of around  1.5 million in the 10x buffer, and 500-800k in the 1x buffer.  I don't really remember what the salt water was.

It was never consistent really and seemed to fluctuate sometimes, and other times fluctuate a lot less, and stay in a consistent range.  Honestly no idea what to take away from it...not a nice solid # like a regular resistor would be.

[Dakota Hamill]

I don't even know what thread is what anymore.

Anyway, here are 3 marker dyes @ 1 min, 20 min, 60 min.

The yellow dye moved about 1 cm in one hour. @ 120V and 100mA 

http://imgur.com/nmG9J5j

Tomorrow I'll soak the paper in a dilute GelGreen solution then try running DNA with a methylene blue loading dye, and spend the remainder of the weekend trying to find a UV light to image it with.  Might have to go to Laser Quest or a rave to find a black light to see the migration.  We'll see how far it gets

[Nathan McCorkle]

Is the water level above the top glass slide? If so you're using a ton
more current than needed. The olde-tyme drawings of paper
electrophoresis setups were triangular, with two separate buffer tanks
connected only by the wet paper:
http://www.funsci.com/fun3_en/exper1/exper1_21.gif

On Thu, Jan 31, 2013 at 6:40 PM, Dakota Hamill <dko...@gmail.com> wrote:
> http://imgur.com/a/t1SEv
>
> Re-doing the experiment but with coffee filter paper, and a real gel box,
> and a real power supply. As you can tell from the first picture, the chem
> filter paper I have allows the dye to disperse way too fast, as it should,
> since it's supposed to let solvents pass through it quickly. Sebastian
> seemed to have decent sounding results using the brown coffee filter paper,
> so we'll see how it works with the dyes.
>
> I tried to reduce pouring turbulence too much when pouring in the buffer
> but, was unavoidable when the solvent front rushed through the slide
> sandwhiches. Running at 120V 100mA or there abouts.
>
> There is already noticeable migration of one of the components of the
> markers to one direction---anyone fancy a guess? You'll win the Marker
> Migration badge of honor.

From what I've been reading today, this might depend on the pH, so
what's your buffer???

:)

>
> I'll try to post a video later if you want to experience the most intense
> marker dye coffee filter electrophoresis you've ever seen. Blow your mind
> if it was in 3D.

Cool!

--
-Nathan

[Dakota Hamill]

Hmm cool old picture, never thought of letting the ends hang down.  Might have to give it a try as it would work well in a gel box still, just cut the paper long and let the ends droop down into the wells.  I did cover the top of slides by a few milimeters of buffer.  It was running in TAE so ~pH 8.  I know yellow dyes are generally negatively charged just from looking at a few of their structures for an old project, but what dyes they use in food coloring vs markers I don't know.  Maybe it isn't that different?

If it took a small dye molecule that long to migrate, I can only imagine how long some DNA might take.  I think I have some 650 and 1kb PCR products left over as well as genomic DNA From a fungi so I'll see if I can do 3 more runs later, but I probably won't be able to visualize the stain unless I get a UV light tonight.

[Jeswin]

On Thu, Jan 31, 2013 at 9:44 PM, Dakota Hamill <dko...@gmail.com> wrote:
> Well I cut 3 strips of filter paper and put them on glass slides. Soaked
> one in salt water, one in 1x TAE, one in 10x TAE.
>
> All read way too high on the multimeter for resistance until I got to the
> 200k and 2 million Ohm setting.
>

Maybe there are different buffers for paper electrophoresis?

[John Griessen]

On 02/01/2013 08:00 AM, Dakota Hamill wrote:
> work well in a gel box still, just cut the paper long and let the ends droop down into the wells. I did cover the top of slides
> by a few milimeters of buffer.

That gave the current easy ways to go around your strip.

It was running in TAE so ~pH 8.

Please tell Ohms from one well of buffer to the other wetted with your chosen buffer
and with the electrodes you use.

Then I can figure power, heat, volts needed to get more, etc.

[Dakota Hamill]

Ok the next time I do a run with dye/DNA I'll try to get better measurements, and i'll leave the high point of the gel box dry with only the wet paper as the path for current to travel in.

[Sebastian S. Cocioba]

So if this works you could do electrophoresis in two beakers and a power supply with a filter strip in the middle? This little curiosity seems to have some serious potential. I'll keep trying on my end and see if I can get some DNA bands. Maybe if we dip the strip into the dna/stain mix you could obtain a uniform front and at least remove the the radial expansion of the dye at the beginning...if of course there is any to begin with. 

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

On Feb 1, 2013, at 3:07 PM, Dakota Hamill <dko...@gmail.com> wrote:

Ok the next time I do a run with dye/DNA I'll try to get better measurements, and i'll leave the high point of the gel box dry with only the wet paper as the path for current to travel in.

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[Nathan McCorkle]

On Fri, Feb 1, 2013 at 12:48 PM, Sebastian S. Cocioba
<scoc...@gmail.com> wrote:
> So if this works you could do electrophoresis in two beakers and a power
> supply with a filter strip in the middle?

Yep

--
-Nathan

[Nathan McCorkle]

On Wed, Jan 30, 2013 at 7:36 AM, John Griessen <jo...@industromatic.com> wrote:
> On 01/30/2013 07:48 AM, Dakota Hamill wrote:
>>
>> Trying to think of other ways to seal off a narrow channel, to control the
>> initial dispersion as you said, but keep open a running
>> lane for the DNA or dye to migrate.
>
>
> It seems to me that ordinary filter papers wick rapidly, making wide blurs
> out of any sample put on them.
>
> If you coated filter paper with agarose then dried it be convenient paper
> again, it might
> perform more like agarose -- low dispersion. If drying somehow ruins
> it,then store
> it wet refrigerated/frozen and you'd get the agarose narrow dispersion
> benefits with a small amount and the convenience of paper.
>
> It might pay to try different papers. Clay coated papers for photo printing
> might be good.
> Reading about TLC made me try searching for silica gel paper and it exists:
> http://www.fishersci.com/ecomm/servlet/fsproductdetail_10652_785049__-1_0
> and is thick, which is good for electrophoresis.
>

I just so happen to have about a gallon of silica powder with a 254nm
excited UV fluorophore (so your compound creates a dark spot) and some
filter paper... I don't have any ladder though, and only methylene
blue stain right now.

https://picasaweb.google.com/lh/photo/ZoqH0q56h_bFbO-lXVqVF-1Smq-NzwwjGgUaj45-NKQ?feat=directlink


--
-Nathan

[John Griessen]

On 02/01/2013 03:00 PM, Nathan McCorkle wrote:
> I just so happen to have about a gallon of silica powder

I ran across a description of thin layer chromatography saying
silica is one good stationary medium for low dispersion and in another place read that it
can be bonded to paper with gypsum, (plaster of paris) to make chromatography
paper.

[Nathan McCorkle]

I bought it to coat glass plates for TLC, I wonder if the DNA would even
get through the silica if you coated paper...

--
-Nathan

[Sebastian S. Cocioba]

If given the proper conditions the DNA would bind and be stuck. As it travels it may shear. Since electrophoresis applies a constant force onto the DNA, it seems that it would pull it to shreds. I believe someone mentioned this exact idea in a previous post.

Sebastian S Cocioba
CEO & Founder
New York Botanics, LLC

Sent via Mobile E-Mail

[Nathan McCorkle]

On Fri, Feb 1, 2013 at 3:55 PM, Sebastian S. Cocioba <scoc...@gmail.com> wrote:
> If given the proper conditions the DNA would bind and be stuck. As it travels it may shear. Since electrophoresis applies a constant force onto the DNA, it seems that it would pull it to shreds. I believe someone mentioned this exact idea in a previous post.

Wait, why would it shred? I used to use silica spin-columns, they
didn't shred the DNA, though varied salt and pH buffers allow
selective binding/unbinding to allow purification of certain size DNAs


--
-Nathan

[Sebastian Cocioba]

My thinking is in the case where the dna is partially bound and is being pulled via its charge across the strip. Could the TAE cause binding? If it does not then it should be fine like you mentioned. The G force of a centrifuge is, i think, much higher than the pull of DNA towards the positive pole so it wont matter. I'm not sure why I thought it would be torn up. Could you think of anything going wrong with using silica in this scenario? 

[Eugen Leitl]

If you're looking at chromatography, just order some
commercial TLC plates, which also come in fluorescent.

http://www.chem-ilp.net/labTechniques/TLC.htm

[Cathal Garvey]

I think that heavily depends on the buffer and pH: under some
conditions, DNA won't have any affinity for Silica, under other
conditions it'll bind strongly.

Spin columns do nick DNA, but I don't think that's due to partial
binding. I've been told (no references, mind) that the mechanism of
DNA:Silica adhesion is through ion-masking of the DNA and Silica charge;
DNA isn't directly bound to silica, but rather ions bind to the backbone
and then bind to the silica. I would imagine that this means partially
bound DNA would "zip" to a completely bound state pretty quickly, but
who knows?

On 02/02/13 00:44, Sebastian Cocioba wrote:
> My thinking is in the case where the dna is partially bound and is being
> pulled via its charge across the strip. Could the TAE cause binding? If
> it does not then it should be fine like you mentioned. The G force of a
> centrifuge is, i think, much higher than the pull of DNA towards the
> positive pole so it wont matter. I'm not sure why I thought it would be
> torn up. Could you think of anything going wrong with using silica in
> this scenario?
>
> On Friday, February 1, 2013, Nathan McCorkle wrote:
>
> On Fri, Feb 1, 2013 at 3:55 PM, Sebastian S. Cocioba
> <scoc...@gmail.com <javascript:;>> wrote:
> > If given the proper conditions the DNA would bind and be stuck. As
> it travels it may shear. Since electrophoresis applies a constant
> force onto the DNA, it seems that it would pull it to shreds. I
> believe someone mentioned this exact idea in a previous post.
>
> Wait, why would it shred? I used to use silica spin-columns, they
> didn't shred the DNA, though varied salt and pH buffers allow
> selective binding/unbinding to allow purification of certain size DNAs
>
>
> --
> -Nathan
>
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[Nathan McCorkle]

On 01/30/2013 07:48 AM, Dakota Hamill wrote:
>
> Trying to think of other ways to seal off a narrow channel, to control the
> initial dispersion as you said, but keep open a running
> lane for the DNA or dye to migrate.

Dakota, not too far off from what this paper did:
Electrochemical Detection for Paper-Based Microfluidics
http://diyhpl.us/~bryan/papers2/paperbot/Electrochemical%20Detection%20for%20Paper-Based%20Microfluidics.pdf

"
Preparation of Paper-Based Microfluidic Devices. Photolithography was
used to pattern Whatman filter paper 1 according
to previously reported methods.
8,12
Briefly, SU-8 3025 photoresist
was poured on the center of the paper and spread over the paper
using a spin-coater. The photoresist-covered paper was baked at
95 °C for ∼10 min. The paper was then covered with transparency
film photomask generated using a standard laser printer and
irradiated with a UV lamp.
"


--
-Nathan

[Dakota Hamill]

Cool  thanks, I'll check it out tomorrow.  I went down to run some PCR and gels today, and wanted to do another paper electrophoresis run with actual DNA today because I got a gel imager....two actually.  The Blue LED strip works fine, but I don't have an orange filter to see the GelGreen, and the weird little UV thing I got seems to not turn on unless you have the more expensive half of the gel imager.  It's one of those visualize as you run gel doc things, and you plug in a gel cartridge and run your gel and at any point can hit a button and turn on the UV Under neath.  It needs an 18V DC at 1.1 A, and I plugged in a 20V capable of 3 A and it still didn't light up so...am thinking the gel cartridge connection needs to be made before it allows the light to turn on, or else it is broken, which could be very possible.   

[Simon Quellen Field]

To make the procedure more DIY friendly, consider using the laser printer toner as the hydrophobic coating.

If the paper is thin, it should soak right through under the laser printer heater.

-----

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