gel electrophoresis vs liquid chromatography?

[GO]

I am lurking here for a while since I have a very tangential experience with bio. My phd is in nano but biohacking looks very interesting and promising. Anyway I was hoping that the community can help me with some questions.

For example, I am confused why is gel electrophoresis so popular compared to liquid choromatography. One constantly needs to buy agarose and visually label dna (have a UV imager) while the same column would work for a long time. We can also co-pass a already known strands with some fluoro or other label that can be detected at wavelength other than 260 nm (this would serve as a ladder in electrophoresis).  Also its more difficult to collect DNA later in gel compared to HPLC. Liquid chromatography can be used to detect other things as well (not just charged DNA) so it's even more useful for DIY people. 

What bothers me is the constant need of supply chain for doing DIYbio compared to computer revolution. I would love to simplify detection (at least) to have minimum of consumables possible. Any ideas?

 

[SC]

Hi GO,

 

I think electrophoresis is normally favored over HPLC is that the equipment is cheaper.   In my experience HPLC has a steeper learning curve and requires occaisonal repairs.  A gel electeophoresis unit rarely needs a repair, just a rinsing out.

 

Stacy

[John Griessen]

On 02/18/2015 01:57 PM, GO wrote:
> I would love to simplify detection (at least) to have minimum of consumables possible. Any ideas?

Perhaps electrophoresis is just a hangover from what was simple and possible.

The gap now is several things:
1. That pressure plumbing requires skill, or special engineered precise fittings for connections
to columns, and the pipping is skinny tubes that can become dirty and be a difficult
cleaning/decontamination job, or an expensive replacement job.
2. That spectrometers or monochrome detectors need precise time stamping to be able
to use the resulting chromatography delay. That means a dedicated computer system...
3. Costs.

It's high time to reduce the costs and the controllers to handle the precise data logging are here too.

For controlling this kind of thing, I like micropython with an ethernet port, but a Black Swift linux board
with wifi instead could be good. Both also have USB, but ethernet and a web server could be nice.

So, if you used a black swift, the linux computer with wifi part is just $25. Add sensors, IO, relay drivers
and secured code-->viable product affordable to DIY/low budget folk.

It also could be a reason to have 6 LC machine setups in a lab instead of just one. Each could have
a certain type of column in it, reducing contamination events since it stays
assembled waiting for use all the time.

[SC]

I've never collected a DNA band from HPLC.  How is it done?

[Dakota Hamill]

The only time I've ever seen an HPLC used for DNA is purification of short oligonucleotides like primers for PCR.  Awesomely enough (is that a word? No, but I'll use it.) it was a Waters 600E which looks like its from the 60's and was part of NASA's command launch console for the first moon missions.  In fact, it was so old the UV detector simply read as a needle that went up and down, not even an LED display.  The tech would stand there and collect the fraction by hand until the needle dropped down, then stop.

I know that company in Canada that does magnetic bead based DNA assembly uses it for purification of their specific oligos.  Genomikon I think they are called.  

I've never heard of large pieces of DNA being purified by HPLC, I imagine the combination of the pressure and column/silica might shear it?  Depends on the stationary phase though I suppose, but I've never heard of anything besides oligos being done by HPLC.  A little bit of refined sea-weed and a DNA stain and you're good to go in 20 minutes.  

There are capillary electrophoresis machines which I think can separate DNA, and perhaps even fraction collect, but I've never been around them on a regular basis.

On Wed, Feb 18, 2015 at 8:59 PM, 'SC' via DIYbio <diy...@googlegroups.com> wrote:

I've never collected a DNA band from HPLC.  How is it done?

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[Nathan McCorkle]

On Wed, Feb 18, 2015 at 11:57 AM, GO <gobi...@gmail.com> wrote:

I am lurking here for a while since I have a very tangential experience with bio. My phd is in nano but biohacking looks very interesting and promising. Anyway I was hoping that the community can help me with some questions.

For example, I am confused why is gel electrophoresis so popular compared to liquid choromatography.

If you consider [silica] spin-columns as being a form of liquid chromatography (they are), then I'd wager that LC in this sense is as popular these days and possibly utilized /more/ often in day-to-day lab operations.

 

One constantly needs to buy agarose and visually label dna (have a UV imager) while the same column would work for a long time.

With common gel electrophoresis, you essentially get a new column each time, so you don't have to think so much about rinsing and carryover from previous experiments. You certainly /could/ continue to use a gel-electrophoresis gel and I bet you'd get a long life out of a given gel. I think you'd still need to refresh the buffer as often.

So one reason could be that schools/industry have enough $$$ to use a new gel each time, and DIYers don't run gels often enough to benefit (because maybe in the down-time the buffer/gel starts to grow something).

 

We can also co-pass a already known strands with some fluoro or other label that can be detected at wavelength other than 260 nm (this would serve as a ladder in electrophoresis).  Also its more difficult to collect DNA later in gel compared to HPLC.

Well yeah, but the whole instrument itself is much more complex than cutting your section of gel and grabbing a spin-column or a piece of dialysis tubing... so you could think that gels are super-low entry cost, higher per-run... but for the cost you get simplicity, and it works. When a gel breaks, you just whip up a new one... if you break a column or an HPLC/LC controller, most lab-tech types won't be able to do anything. Even with imaging a gel, the common user has a few options they can try out (use a blacklight or UV spot light, use a visible gel dye, use 405nm LEDs, etc). With any kind of 'instrument' that user is stuck, especially if it's an expensive one (and they aren't in an institution with a service contract, or some 70-year old polymath who never graduated high-school but 'started with radios in the army').

 

Liquid chromatography can be used to detect other things as well (not just charged DNA) so it's even more useful for DIY people. 

What bothers me is the constant need of supply chain for doing DIYbio compared to computer revolution.

In what sense though? Computers need minerals, floppy disks and thumbdrives go bad over time, need fed electricity constantly. Things are just different.

 

I would love to simplify detection (at least) to have minimum of consumables possible. Any ideas?

 

Microfluidics is a big idea (haha, didn't intend the pun). You still need some pumps though, even for a capillary gel electrophoresis, I tried the easiest idea of cutting a microchannel and scraping liquid agarose gel over it, then slapping a microscope slide on top and it didn't work, at that scale the channel surface ('electrics') was repulsive enough such that pretty much no gel ended up in the channel.

The detector is another thing, but essentially, there are lots of things that are complex, and the integration of them is complex too. Then you have to work on cost of materials and labor to build the thing, you have to consider user-experience and making it better than whatever convoluted/expensive procedure the user is already using. And then make sure the cost is also acceptable.

Building an HPLC isn't something hard to do for a group like DIYbio, but doing it well and such that it is useful/reproducible outside the group is harder. Getting all the key players to work for months with no pay is also a factor. Plenty if not all the people on this list are willing to type messages to the world for no pay, which means they're likely to devote some time for free elsewhere... but getting everyone's schedule to line up is tough. Science and engineering is tough, tougher than code because you have to leave your keyboard, you have to think about the logic that makes up the real world, not just the logic of whatever the programming language supports. It's much wider and deeper, move nuanced.

If you have some ideas, let's hear them!

I'm up to work on microfluidic gel electrophoresis capillaries, if other folks were willing to help with choosing things like what port/connector to use on the microfluidic portion of the system and other parts of the system too.

[GO]

Thanks you all, people! It certainly helps to discuss this. By the way, sorry for the bad grammar, I was in a hurry.

I agree that huge pressures in HPLC is a drawback. I totally missed but obviously one can use the gel several times and even elute at the end so basically with few modifications it is the same system. For example, if one could put a detector at the end of the gel and make collection wells at the end, it would be the same just simpler because voltage would drive it instead of pressure. And we wouldn't need labeling if detector is for 260nm. 

>Stacy: I agree, HPLC is prone to more problems. Collection in HPLC is very easy, just need to put your tube at the end of the tubing (any point after the column and detector) and have some time estimate of distance between the detector and the tubing end. The sample would be diluted though.  

>John: Pressure is a bummer but I don't agree for timing. The typical run is not so fast and timing is not a problem IMO but as for electrophoresis you would need dedicated system (like power supply). Just since you are using eyes for electrophoresis, detection is easier. 

>Dakota: Good point about the size. I looked it up, and they can do bigger dna but with the ion exchange column protocol so it is more complicated.

> Nathan: Unfortunately I don't have any good ideas right now but my goal would be to make detection of biomolecules so easy that average Joe can do with with one device at home and no consumables. I guess the kits are the closest thing for that but the fact that you can't run the same thing repeatedly is a major setback. You need to purchase kit or stick (for drug or pregnancy detection) every time. Actually I need to take a look at those diabetics sensors/dispensers. 

Regarding the agarose microchannels, I made some microstamps with agarose so my guess is that charge is not your problem but more likely that you didn't keep the whole thing hot when you tried to get agarose inside. If it is gelled than it breaks easily. I can send you the images that I took and we can discuss. I am interested in that project but did you search the papers (I am pretty sure it was done before).

[Dakota Hamill]

You don't need to elute it off the gel, you can just excise it with a razor blade and prep your DNA from it.  That's a common practice.

As for your wells on a gel idea, a local company here has done that.  Sage science with the Pippin Prep.

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[GO]

Nathan, here is the image of agarose on glass. I think that the stripes are about 10 microns thick. It was done by pressing a PDMS stamp on a hot glass slide with agarose. I was interested in nanoparticles not stripes so I didn't experiment with this much but I was able to make squares of agaraose. The problem was the background or residual. I would probably need some flouropolymer to reduce that.  

[Dakota Hamill]

You're on the right track of thinking though.  I don't know how well the Pippin Prep works, I've heard mixed reviews.  There is always room for improvement.  There was a lab someone showed me at Boston University that made a pretty cheap (I think) fully functional "PCR machine on a chip" the size of a glass slide.  I think it works by cycling fluid over pre-heated areas that denature, anneal, elongate, etc.  Being able to do that, then 30 cycles later send it out another channel directly into a "gel on a chip" or something else would be cool.  

[Jeswin]

Are you talking about a capillary filled with agarose as a means of
eluting DNA products? The closest thing I know where you can collect
your DNA of interest without cutting is using the Invitrogen e-gel
cassettes. It's a pre-made gel formed in a plastic square with
electrodes at each end. You place this on a special instrument that
applies voltage. The cassette is not immersed in any running buffer
and at the middle of it is open lanes which you fill with a elution
liquid. When your sample reaches the collection lane, simply pipette
out the liquid. It's pretty neat, but quiet expensive compared to
regular agarose gel electrophoresis.

> https://groups.google.com/d/msgid/diybio/CAGdeWmSwetBYc_Wu%3DhM6Y711yvueADL0ZrGS%3Dz8qtycN9NU_7w%40mail.gmail.com.

>
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[John Griessen]

On 02/19/2015 01:59 AM, Nathan McCorkle wrote:
>
> I'm up to work on microfluidic gel electrophoresis capillaries, if other folks were willing to help with choosing things like what
> port/connector to use on the microfluidic portion of the system and other parts of the system too.

What do you think of that polyamide pcb I sent you as microchannel fabbing tech? It's a material that
can be bonded, and I think it is fairly inert for chemistry reactions and purity...and its a fine insulator
if thinking of applying Volts. The channel size seems like around 50 micron, but I did not put a ruler to it yet.

On 02/19/2015 09:32 AM, Dakota Hamill wrote:> fully functional "PCR machine on a chip" the size of a glass slide. I think it

works by cycling fluid over pre-heated areas that
> denature, anneal, elongate, etc. Being able to do that, then 30 cycles later send it out another channel directly into a "gel on
> a chip" or something else would be cool.

Simple yet tiny valves and pumps are part of what's needed.

[Dakota Hamill]

I was working with someone who mentioned it to me and showed me a few pictures online, I think these are from that time, though it was a year ago so I don't recall exactly.

http://www.medgadget.com/2012/04/new-microfluidic-chip-for-low-cost-rapid-testing-of-influenza-strains.html

http://www.bu.edu/klapperich/

I'm 99% sure that was the lab it came from

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[GO]

Looks neat, thanks! My problem with this and with the others is that you need reagents. I will take a closer look though, not sure where they put the reagents, maybe I am wrong.

On Thursday, February 19, 2015 at 10:59:07 AM UTC-6, Dakota wrote:

I was working with someone who mentioned it to me and showed me a few pictures online, I think these are from that time, though it was a year ago so I don't recall exactly.

http://www.medgadget.com/2012/04/new-microfluidic-chip-for-low-cost-rapid-testing-of-influenza-strains.html

http://www.bu.edu/klapperich/

I'm 99% sure that was the lab it came from

On Thu, Feb 19, 2015 at 11:43 AM, John Griessen <jo...@industromatic.com> wrote:

On 02/19/2015 01:59 AM, Nathan McCorkle wrote:

I'm up to work on microfluidic gel electrophoresis capillaries, if other folks were willing to help with choosing things like what
port/connector to use on the microfluidic portion of the system and other parts of the system too.

What do you think of that polyamide pcb I sent you as microchannel fabbing tech?  It's a material that
can be bonded, and I think it is fairly inert for chemistry reactions and purity...and its a fine insulator
if thinking of applying Volts.  The channel size seems like around 50 micron, but I did not put a ruler to it yet.

On 02/19/2015 09:32 AM, Dakota Hamill wrote:> fully functional "PCR machine on a chip" the size of a glass slide.  I think it works by cycling fluid over pre-heated areas that
> denature, anneal, elongate, etc.  Being able to do that, then 30 cycles later send it out another channel directly into a "gel on
> a chip" or something else would be cool.

Simple yet tiny valves and pumps are part of what's needed.

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[GO]

I think that might be it (capillary electrophoresis) what I need. Need to read.

Yes, sounds like Invitrogen e-gel is a reusable thing. I'll take a closer look.

Thanks Phllyj!

[Nathan McCorkle]

On Thu, Feb 19, 2015 at 8:43 AM, John Griessen <jo...@industromatic.com> wrote:

On 02/19/2015 01:59 AM, Nathan McCorkle wrote:

I'm up to work on microfluidic gel electrophoresis capillaries, if other folks were willing to help with choosing things like what
port/connector to use on the microfluidic portion of the system and other parts of the system too.

What do you think of that polyamide pcb I sent you as microchannel fabbing tech? 

It's a material that
can be bonded, and I think it is fairly inert for chemistry reactions and purity...

I haven't looked into the chemical compatibility yet, but I do wonder if they sell them without being sandwiched together, or what other options they might provide. Would the channel walls be metal or polyimide? How would the void/channel space be realized, etc. Also, if 50 microns is the lower limit, I wonder what the cost of these in small-volumes is compared to fabbing a master impression stamp, then stamping some silicone.

For something like gel electrophoresis, the chemicals aren't too harsh generally, so chemical compatibility seems like it would probably be fine.

Connecting the end of a sandwich might be somewhat challenging (since the connection would have to support some pressure to pump in the agarose), but grabbing the end and pulling it into a silicone slab with a hole in it might work for connecting the capillary up to a macro-connector.

[Nathan McCorkle]

On Thu, Feb 19, 2015 at 7:31 AM, GO <gobi...@gmail.com> wrote:

Nathan, here is the image of agarose on glass.

Neat! I didn't consider that the agarose might have been too cool. Here is an image of the device I tried to place agarose into:

http://reportermagarchive.com/files/cache/2803_maxsize_800_800.jpg

I still have it actually, so I could try again with it. It was a piece of silicon with PDMS spin-coated, then laser-etched to form channels (and these channels were investigated with a 2D interferometer, showing the surface was quite rough, not unlike swiss cheese). I think we just pipetted or poured agarose over the channel then smashed a microscope slide on top, then tried to electrophorese some DNA through it. There were a few problems, like the ends didn't have tall reservoirs for buffer, so maybe the buffer boiled away and messed up electrophoresis, but it seemed like the agarose didn't make it into the channel from what we could tell. It's been 3 or 4 years though, so a bit hard to remember. I do remember coming away thinking I needed the top cover sealed so I could inject agarose. Maybe plasma treating the silicone beforehand would have alleviated this, not sure.

 

I think that the stripes are about 10 microns thick. It was done by pressing a PDMS stamp on a hot glass slide with agarose. I was interested in nanoparticles not stripes so I didn't experiment with this much but I was able to make squares of agaraose. The problem was the background or residual. I would probably need some flouropolymer to reduce that.  

I wonder if those agarose 'strips' could be used as-is, or with little modification.... add a dimple at one end to place the sample, and run it in a very humid chamber to keep the agarose wet... maybe with some buffer reservoirs at each end.

[John Griessen]

[John Griessen]

On 02/19/2015 01:28 PM, Nathan McCorkle wrote:
> Connecting the end of a sandwich might be somewhat challenging (since the connection would have to support some pressure to pump
> in the agarose),

Oh, connecting right at a cut edge is not necessarily the way to get it done. This is fabbing from low cost films in
clean conditions, and bonding them together. A connection can probably be made to a hole in a film by some larger structure
just like a Si chip was bonded to the polyamide tape on that circuit example I sent you Nathan. Looks like tiny bead of epoxy.

On 02/19/2015 01:36 PM, Nathan McCorkle wrote:> I wonder if those agarose 'strips' could be used as-is, or with little
modification.... add a dimple at one end to place the
> sample, and run it in a very humid chamber to keep the agarose wet... maybe with some buffer reservoirs at each end.

Like that idea. That could become a low cost prepared cartridge sealed with tape, ready for one use after pulling out a tab.

I like the way it avoids attempting to push "jello" through a "super skinny straw", (lukewarm agarose : microchannel).

[Nathan McCorkle]

I just did HPLC today on caffeine and it was pretty easy... I was
checking column prices last week on ebay and they seemed like around
$50 for used... but what about new?

What are the ideal DNA columns? Is there an easy/cheaper DIY
compromise/substitute available? (maybe silica?)

On Wed, Feb 18, 2015 at 11:57 AM, GO <gobi...@gmail.com> wrote:

> We can also co-pass a already known strands with some fluoro or other
> label that can be detected at wavelength other than 260 nm (this would serve
> as a ladder in electrophoresis).

Concern might be that the dye dislocates during chromatography...

> Also its more difficult to collect DNA
> later in gel compared to HPLC. Liquid chromatography can be used to detect
> other things as well (not just charged DNA) so it's even more useful for DIY
> people.

I didn't collect elutions, but I think I would have just had to figure
out the lag time between the detector and the end of the waste tube.
It certainly would have been easy. The one problem I could see is that
while the sample loop was only 100uL, it was recommended to flush with
1000uL (to clean out any trace of previous sample) and I was using
more like 2 or 3 mL since we had plenty. So for people with only 50uL
or even 500uL, we need to figure something out... as a gel can be
loaded with something like 10-20uL. I am not sure if diluting it to
1000uL would be smart or not... I am sure the required volume could be
decreased, but there is still the flush volume to prevent
cross-contamination... I guess this could be switched from sample
being used to wash/rinse to DNA buffer.

I think the high-pressure generation and injector valve is the hard
part of this. Maybe a good reason to go after capillary (or capillary
gel electrophoresis) electrophoresis instead... no pressure to worry
about, so adding sample shouldn't be much different for end-users used
to loading gel wells.

[GO]

I used the columns that you can fill yourself with the HPLC machine. Don't know how much does it cost though. For DNA and some bigger stuff, I think I used Sepharose and Agilent used to have the whole range that you can choose from depending on what resolution you need. Regarding the dye labeling, I actually meant the covalently modified DNA. I used that before and of course that is stable; I admit that would be expensive though. Typically people run stuff sequentially and compare times but there is a variation from run to run, so it is more reliable and faster if you can do it in just one run.

Huge pressure is a bummer for HPLC. If we could do it in gas phase, it would be lower pressure for sure, less adsorption to the column material too and possibly faster (that would reduce the resolution though). I know that they gas chromatography is used for volatile stuff but I am puzzled why it could make it work for lyophilized stuff for example.    

Where did you get HPLC machine? They look pretty expensive on Ebay.