DIY Microfluidic valves - Anyone know how to homebrew one?
ByoWired
I'm interested in sorting cells but I would like to prevent the cells
from getting contaminated and vice versa, so I don't think I want to
try the kind of cell sorter that electrostatically separates droplets
in the air. I've been trying to find a way to separate cells using
DIY microfluidics, but I'm not finding anything suggesting that such
tiny valves could be made by a DIYer. Basically I just need a simple
Y-shape in tubing that has an inside diameter of about 50 to 100
micrometers. Somehow the cells have to be guided down one branch of
the Y or the other. Solenoid actuators operate with a reaction time
of roughly 20 milliseconds, which means a sorter of this kind would
run fairly slow compared to a traditional flow cytometer (in air) cell
sorter. Piezo actuators can go much faster but they cost a fortune,
especially if they have to move distances greater than about 15
micrometers. I know there are all kinds of fancy electrowetting
shuttling systems and so forth, but I don't see how those can be done
inexpensively in grand DIY-style.
Anyone have any suggestions? insights? hallucinations?
thanks,
Mark
Nathan McCorkle
cells (all off shelf), $1000-$1500 easily
I can get you brands and models of positive displacement pumps
(stepper driven, 50-1000 or so ul per full stroke of the pump
depending on model, so each step can me nanoliters) made of some sort
of zirconia so they'll stay accurate for a long time, they hook to a
valve that is a rocker valve, which would then go to your flow cell
setup... could make a flow cytometer this way which would be at least
$3000 including detectors and lasers and flow setup
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Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
ByoWired
On Oct 2, 4:29 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> DIY as a kit type project would cost, for pumps and valves and flow
> cells (all off shelf), $1000-$1500 easily
rocker valve? Is there a particular brand or model that has been
developed for, or is adaptable to, microfluidics? It seems to me that
any kind of valve solution would have to be a pincher type, since dead
space volume in any other kind of valve would defeat the selection
process.
I've got no problem making pumps. Nor do I have a problem with the
detection electronics that can identify a wanted cell from an unwanted
cell. Where I'm stuck is at the branching fork of the sorting
process, the Y in the tubing that I mentioned earlier. One solution
is to somehow use tiny flexible tubing and develop pinch valves that
pinch/unpinch the different branches of the Y depending on whether a
cell is wanted or not. But how to do that DIY?
Nathan McCorkle
http://www.idex-hs.com/product-families/2/Valves-.aspx
pinching will cause an effect in the flow... volume will be displaced
as the tube changes size... to get around this companies (maybe Idex,
but i can ask a guy at work) also make valves that actuate by rotating
a plate (or two), with the fluid vias connecting in one plate
orientation and not the other.
a rocker is just that, an arm balanced on an axis, in one extreme of
being tipped to a side, it is open, when the arm tilts it effectively
pinches off the fluid. I'm not sure how well sterile these can be
maintained, if not by chemical means, then I don't know... doubtful
they can be sterilized...
a pincher is a good idea though... let me know what you start thinking
after you read this and the idex site
ByoWired
On Oct 2, 11:06 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> these guys make the valves I'm referring to:http://www.idex-hs.com/product-families/2/Valves-.aspx
>
> pinching will cause an effect in the flow... volume will be displaced
> as the tube changes size...
It's true that pinching a tube will do bizarre things to the flow, but
if you can time the pinching of one branch of the Y with the
unpinching of the other branch, it seems to me that the flow in the
main stream wouldn't be affected too terribly much(???). It might be
tricky getting the two actions to balance out, however. Just not
sure. There is also the big question of cyclic fatigue. Flow
cytometer cell sorters that work in air don't use moving parts to sort
the droplets - it's purely electronic, so sorting through millions of
cells causes no wear and tear. However, having mechanical valves that
actually have to move anything back and forth raises the question:
just how many cycles can they move before they fail? And if a tube
pincher is used, just how many cycles can the tubing take before it
starts to crack, etc.?
Right now I've got way more questions than answers.
Thanks again,
Mark
Cathal Garvey
Is there a way to make solid state microfluidic pumps using the electric current method I've heard featured in "The Hunt for Red October"? Apply current across your microfluidic stream with a perpendicular magnetic field across the other cross-flow axis, and faraday sez the fluid should move if it conducts any current. If you can get such a small, solid state pump running on either leg of your junction, then rather than 'pushing' your fluid down the right path you can 'pull'.
I don't know if this works in real life, but it'd be a solid state method requiring few parts.
Another option might be to constrict the tubing from outside with nitinol or an actuated hose clamp
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On 3 Oct 2010 05:29, "ByoWired" <byow...@gmail.com> wrote:
On Oct 2, 11:06 pm, Nathan McCorkle <nmz...@gmail.com> wrote:
> these guys make the valves I'm ref...
Nathan, thanks for the link to the Idex website.
It's true that pinching a tube will do bizarre things to the flow, but
if you can time the pinching of one branch of the Y with the
unpinching of the other branch, it seems to me that the flow in the
main stream wouldn't be affected too terribly much(???). It might be
tricky getting the two actions to balance out, however. Just not
sure. There is also the big question of cyclic fatigue. Flow
cytometer cell sorters that work in air don't use moving parts to sort
the droplets - it's purely electronic, so sorting through millions of
cells causes no wear and tear. However, having mechanical valves that
actually have to move anything back and forth raises the question:
just how many cycles can they move before they fail? And if a tube
pincher is used, just how many cycles can the tubing take before it
starts to crack, etc.?
Right now I've got way more questions than answers.
Thanks again,
Mark
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ByoWired
On Oct 3, 10:09 am, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Is there a way to make solid state microfluidic pumps using the electric
> current method I've heard featured in "The Hunt for Red October"?
nanotechnologies. But there seems to be two problems from a DIY
perspective. First, to make such tiny circuits takes fairly expensive
equipment. Second, some of the techniques like electrowetting and so
forth (which can be done using printed circuit boards accessible to
DIYers) require voltages so high that electrical arcing gets to be a
problem and living cells might get burned/electrolyzed, etc. by the
sheer magnitude of the voltage. A DIY approach is challenging because
it requires a scale at which a normal human being can deal with the
mechanics (in other words, it's hard to make nanoscopic devices with a
Dremel, etc.).
The problem with nitinol wire is that it would be excruciatingly slow
compared to the desired rates. And if you're talking about the screw-
type hose clamp, I'm afraid those, too, would be far too slow.
Generally speaking, microfluidics is slow compared to the in-air cell
sorters. So I guess I've got a battle on two fronts: coming up with a
new, faster way of doing things and having it be a DIY solution, too.
I am awaiting an epiphany.
Thanks Cathal. :)
Cathal Garvey
Well, what I was thinking of would amount to some wires and small magnets embedded in a gel-cast tube. The small gap between the electrodes might, for certain conductivities, require only a few volts (arduino?) to generate a useful current through the fluid, which the embedded magnets would apply a force against.
Casting it might involve suspending the electrodes and magnets around a removable plastic Dowel, and pouring a gel on top. Interfacing this with the rest of your setup might be challenging but if the whole thing were gel-cast it wouldn't be much extra work to do it at-once.
All this spoken by someone who's never done any microfluidics so take it all with a pinch of salt: could be nonsense.
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Sent from my beloved Android phone.
On 3 Oct 2010 16:17, "ByoWired" <byow...@gmail.com> wrote:
On Oct 3, 10:09 am, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Is there a way to make solid s...
Yes, there are all kinds of ways to make fluids move with fancy
nanotechnologies. But there seems to be two problems from a DIY
perspective. First, to make such tiny circuits takes fairly expensive
equipment. Second, some of the techniques like electrowetting and so
forth (which can be done using printed circuit boards accessible to
DIYers) require voltages so high that electrical arcing gets to be a
problem and living cells might get burned/electrolyzed, etc. by the
sheer magnitude of the voltage. A DIY approach is challenging because
it requires a scale at which a normal human being can deal with the
mechanics (in other words, it's hard to make nanoscopic devices with a
Dremel, etc.).
The problem with nitinol wire is that it would be excruciatingly slow
compared to the desired rates. And if you're talking about the screw-
type hose clamp, I'm afraid those, too, would be far too slow.
Generally speaking, microfluidics is slow compared to the in-air cell
sorters. So I guess I've got a battle on two fronts: coming up with a
new, faster way of doing things and having it be a DIY solution, too.
I am awaiting an epiphany.
Thanks Cathal. :)
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ByoWired
On Oct 3, 12:44 pm, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Well, what I was thinking of would amount to some wires and small magnets
> embedded in a gel-cast tube.
a wide variety of things that don't seem like they would apply. Is
there a particular material you think might be good for this? I know
that the standard microfluidics people use Polydimethylsiloxane
(PDMS), but even that is a bit tricky to use, from what little I
understand of it. When making such tiny tubes (ID of about 100
micrometers) I know things get crazy. And to embed things with circa
200 micrometer spacing, might be difficult, too. Anyway, I'm clueless
about casting.
Thanks for the suggestions.
John Griessen
> I'm interested in sorting cells but I would like to prevent the cells
> from getting contaminated and vice versa, so I don't think I want to
> try the kind of cell sorter that electrostatically separates droplets
> in the air. I've been trying to find a way to separate cells using
> DIY microfluidics, but I'm not finding anything suggesting that such
> tiny valves could be made by a DIYer. Basically I just need a simple
> Y-shape in tubing that has an inside diameter of about 50 to 100
> the Y or the other.
.
.
.
Hi, I'm new here, I have experience in power supplies mixed signal chips and
chip and board layout. This makes me think of a way related to pinching:
> I've got no problem making pumps.
> Where I'm stuck is at the branching fork of the sorting
> process, the Y in the tubing . . . . develop pinch valves that
> pinch/unpinch the different branches of the Y depending on whether a
> cell is wanted or not. But how to do that DIY?
Pinching might be problems, and implies a large 1/4 inch tubing scale,
which is not much help. The thought I have is to make a mechanically
or feedback control linked differential pump with smooth control of it's
differential flow via some electronics.
If 50 to 100 um is good for the flowing path that Y's into select and reject paths,
then a likely diameter for the two lines coming from a differential pump is double
the cross section area, so 71 to 141 um.
The hallucination I got is sketched here: http://ecosensory.com/diybio/flow-diff-pump-1.jpg
The key to DIYing this is toseparate the micro from the macro at the tubing fitting
level, where parts is parts and they're cheap. The F1 and F2 in the diagram
are forces that could come from macro sized organic piezo actuators
that are not so stiff and not so strong and inexpensive. B1 and B2 are
ports for some stiffly delivered bleed flow. That can be done full manual
to start by just supplying clean bleed water at much higher pressure than
the sorter has and dropping that through a needle valve to turn it into a flow
that is fairly insensitive to local fluctuations that cause sorting.
If F1 and F2 oppose and supply a volume of water no bigger than the amount
used to separate cells, they can be operated smoothly to shift a cell
one way or the other. The output lines from the sorter need to be long
enough to resist flow so the flows back and forth from F1 and F2 pushing
and pulling affect the sort zone much more than they affect the flow in
the select or rehect lines. The stream of water and cells coming in
needs to be stiffly delivered (pos displacement pump?) also to keep
things always moving in.
If you like this idea, how many of you are there? This could become
a kickstarter project for me to deliver tested, working, and open licensed
for rapid improvement.
John Griessen
Cathal Garvey
Well as I said, I don't do microfluidics (yet?), I was imagining an agarose gel with channels cast into it. I'm not sure how one might easily cast channels and remove the molds after but I'm sure it's doable..
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On 3 Oct 2010 21:18, "ByoWired" <byow...@gmail.com> wrote:
On Oct 3, 12:44 pm, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Well, what I was thinking of w...
What exactly is gel-cast? I tried to google it but I'm coming up with
a wide variety of things that don't seem like they would apply. Is
there a particular material you think might be good for this? I know
that the standard microfluidics people use Polydimethylsiloxane
(PDMS), but even that is a bit tricky to use, from what little I
understand of it. When making such tiny tubes (ID of about 100
micrometers) I know things get crazy. And to embed things with circa
200 micrometer spacing, might be difficult, too. Anyway, I'm clueless
about casting.
Thanks for the suggestions.
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John Griessen
> Well as I said, I don't do microfluidics (yet?), I was imagining an agarose gel with channels cast into it. I'm not sure how one
> might easily cast channels and remove the molds after but I'm sure it's doable..
But would it last? And it wouldn't autoclave.
Glass is the thing for labware, even if it's micro. Cover slips and slides could be etched
and silicate bonded to make channels that lead out to a distance big enough to attach macro
sized tubing. Just thought of a way to strain relief the tubing attachments... use more stacked
and bonded cover slip glass with etched circles to hold small tubing, about 1mm diameter.
When the glass part is cleaned and autoclaved, just plug in the tubing and add glue or
silicone caulk to keep it and seal it. Perhaps the circle edges can be etched to be smooth enough
to seal when a plastic tube is shoved in -- without glues. The shape of a plate of glass with holes
is round edged holes, not sharp/square. They might make a good friction fit with teflon or poly tubing.
The same technique of etched shapes stacked and bonded could make holding tabs for a sensor
for detecting cell presence in the zone, just before swooshing it sideways one way or the other.
John Griessen
ByoWired
that's a very interesting hallucination and there's no reason I can
think of why it wouldn't work. In fact, I think it's somewhat similar
to concepts related to hydrodynamic steering, etc. which have been
tested in expensive microfabrication with reasonable success (though I
think they're still fairly slow compared to cell sorters in air).
There is still the problem of how to manufacture the branching zone,
where the cells encounter the fork in the road. But I recently came
across some parts that are about $20 each that allow fairly small ID
tubing (360 micrometer) to be connected to the "macro" world of Luer
locks, etc. The OD of the tubing isn't insanely small, either. Check
out:
http://www.labsmith.com/microfluidicstable.html
So perhaps the branching fork could be assembled from these T's ???
As for what an "organic piezo" actuator might be, are you talking
about PDVF? That's a very interesting idea. Do you know of any off-
the-shelf items that might possibly perform the magic you speak of?
Thanks for sketching out your ideas on this.
To Cathal,
it never occurred to me to cast something from agarose, and if
somebody wanted a disposable unit I suppose that might be a way to go,
so long as there's a way to make tubes in it. (Can anyone think of a
way to wrangle microbes into eating reasonably controlled
microchannels through agarose? Entice them to follow an electrical
current, eating their way along the shortest path of resistance? I'm
just thinking out loud here.)
thanks again to all for these insightful inputs!
Mark
John Griessen
related to hydrodynamic steering, etc. which have been
> tested in expensive microfabrication with reasonable success (though I
> think they're still fairly slow compared to cell sorters in air).
Very quick compared to pushing at them with a dissecting probe.
I recently came
> across some parts that are about $20 each that allow fairly small ID
> tubing (360 micrometer) to be connected to the "macro" world of Luer
> locks, etc.
Isn't 360 um too big to keep cells in single file?
>
> http://www.labsmith.com/microfluidicstable.html
>
> So perhaps the branching fork could be assembled from these T's ???
The scale of the branching fork needs the low resistance path for pushing
side to side with to be short. Short in the micro scale section of things.
Short as in less than 5 times the width... 250 um up to the point the
paths to the diaphragm pumps widens out to macro size...
That a sideways path is low impedance to flow by
being wider and short is what gives you the possibility of speed. I can't
see making anything very useful out of macro parts. Photolithography
and etching of glass is it. 50 and 71 microns may be tricky. I bet there
are fabs for glass that are not super expensive. 3 mils is a usual process
dimension for circuit board traces in copper, so it is definable with ordinary
darkroom equipment or UV drawing laser printer and then etchable with
the developed photoresist.
To do glass I'm not sure what photoresist is used so the hydrofluoric eats the glass
and not the resist. 3 mils is 76 um... just what you want.
>
> As for what an "organic piezo" actuator might be, are you talking
> about PDVF?
I was wrong about the organic part, the ones I am thinking of:
http://www.piezo.com/prodbm0nav.html
are layered ceramic,
probably PZT, of two or four layers that would be good at quick response
driving of a small diaphragm pump that is macro sized... maybe one cm across.
Mini diaphragm pumps can probably be bought.
You could make them as zones in glass out of cover slip over several layers
of cover slip glass bonded together. Like a drum. The rig the piezo pushers
to push on them.
What do the usual flow cytometer sorters cost? Think there's a market for
inexpensive ones?
John Griessen
Cathal Garvey
Isn't 360 um too big to keep cells in single file?
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Cathal Garvey
Nathan McCorkle
From: sebastien dazy the frog eating cheese <sebasti...@gmail.com>
Date: Sun, Oct 3, 2010 at 3:26 AM
Subject: Re: DIY Microfluidic valves - Anyone know how to homebrew one?
To: Nathan McCorkle <nmz...@gmail.com>
Not sure if it could fit your need but doing a temporary clotting
using ferromagnetic nanoparticules with external permanent magnet
could perhaps be a good valve mimic.
best
sebastien
On 2 oct, 22:29, Nathan McCorkle <nmz...@gmail.com> wrote:
> DIY as a kit type project would cost, for pumps and valves and flow
> cells (all off shelf), $1000-$1500 easily
>
> I can get you brands and models of positive displacement pumps
> (stepper driven, 50-1000 or so ul per full stroke of the pump
> depending on model, so each step can me nanoliters) made of some sort
> of zirconia so they'll stay accurate for a long time, they hook to a
> valve that is a rocker valve, which would then go to your flow cell
> setup... could make a flow cytometer this way which would be at least
> $3000 including detectors and lasers and flow setup
>
> On Fri, Oct 1, 2010 at 11:59 PM, ByoWired <byowi...@gmail.com> wrote:
>
> > I'm interested in sorting cells but I would like to prevent the cells
> > from getting contaminated and vice versa, so I don't think I want to
> > try the kind of cell sorter that electrostatically separates droplets
> > in the air. I've been trying to find a way to separate cells using
> > DIY microfluidics, but I'm not finding anything suggesting that such
> > tiny valves could be made by a DIYer. Basically I just need a simple
> > Y-shape in tubing that has an inside diameter of about 50 to 100
> > micrometers. Somehow the cells have to be guided down one branch of
> > the Y or the other. Solenoid actuators operate with a reaction time
> > of roughly 20 milliseconds, which means a sorter of this kind would
> > run fairly slow compared to a traditional flow cytometer (in air) cell
> > sorter. Piezo actuators can go much faster but they cost a fortune,
> > especially if they have to move distances greater than about 15
> > micrometers. I know there are all kinds of fancy electrowetting
> > shuttling systems and so forth, but I don't see how those can be done
> > inexpensively in grand DIY-style.
>
> > Anyone have any suggestions? insights? hallucinations?
>
> > thanks,
> > Mark
>
> > --
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ByoWired
On Oct 4, 3:27 am, John Griessen <j...@industromatic.com> wrote:
> Isn't 360 um too big to keep cells in single file?
laminar at whatever flow speed you drive it at. I haven't worked out
any Reynolds numbers yet, but hydrodynamic focusing is used to keep
cells centered in regular flow cytometers, so I think it would work
here, too, so long as the Reynolds number stayed low.
>3 mils is a usual process
>
That's interesting.
>
> What do the usual flow cytometer sorters cost? Think there's a market for
> inexpensive ones?
more. The biggest expense is probably the detection equipment - argon
lasers, photomultipliers, high speed electronics, optical filters,
etc. Lab-on-a-chip concepts are out there to make all of that
cheaper, but those people are bumping up against the laws of fluid
physics as it pertains to microfluidics. I'm sure they'll get cheaper
someday, but of course I'm looking for DIY solutions that work "well
enough", not something to compete with the big boys.
To Cathal,
yes, I think your comment about dilution is probably key to a DIY
approach. However, that gets expensive in terms of buffer solution.
I've done some simple calculations that suggest dilutions for larger
ID tubing require something like 5 to 10 liters of buffer. Even if
the buffer can be recirculated somehow, it still needs to be filtered
through very small filters fairly fast, perhaps even sterilized, so
that puts another monkey on my back. The problem with larger tubing
is that you start getting into Reynolds numbers that are too high,
turbulence starts, and you lose all ability to hydrodynamically focus
the fluid, so cells wiggle every which way and your microscopic optics
breaks down. Using lasers then becomes maybe impossible.
Thanks again,
Mark
ByoWired
On Oct 4, 10:04 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> ---------- Forwarded message ----------
> From: sebastien dazy the frog eating cheese <sebastien.d...@gmail.com>
> Date: Sun, Oct 3, 2010 at 3:26 AM
> Subject: Re: DIY Microfluidic valves - Anyone know how to homebrew one?
> To: Nathan McCorkle <nmz...@gmail.com>
>
> Not sure if it could fit your need but doing a temporary clotting
> using ferromagnetic nanoparticules with external permanent magnet
> could perhaps be a good valve mimic.
> best
> sebastien
achieved with that? Any idea about biocompatibility or how to keep
the nanoparticles from being washed away?
thanks,
Mark
Cathal Garvey
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Nathan McCorkle
creates droplets at the nozzle where charge is induced (is that what
you meant by in air??)
> --
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Nathan McCorkle
cells into something like a cement mixer that would act to centrifuge
the cells enough that you can take away excess buffer and recirculate
it. Or maybe to decrease complexity of always on centrifugation, it
could start and stop centrifuging when the liquid level reach a
certain level... pellet cells, suck off fluid, recirculate buffer,
start sorting again
if you were just making a cell sorter, you wouldn't need a gas laser,
i think they're used mainly for flow cytometers because of their
photonic/spectral stability... basically they can give you more
reliable concentration measurements... for just color detection, cheap
solid state lasers should be fine... and if you were driving them with
decent current control circuits (pretty easy for an electrical engi
student) they would not be too bad for concentration measurements
either.
the control system would probably be able to be driven by a beagle
board, its got plenty of power for image processing and such
> --
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John Griessen
> http://en.wikipedia.org/wiki/Magnetohydrodynamic_drive
> This is the concept I was talking about, by the way. In case anyone thought I was mad, apparently it works
Yes, the hunt for red october method, or course.
What is more DIYable is to take this MEMS method and macro size the coil part:
http://books.google.com/books?id=bzxZkEcLo1kC&pg=PA794&lpg=PA794&dq=micro+glass+diaphragm+pump&source=bl&ots=WYTLzXqRgz&sig=5PwcVrMTxESx4HpCGXbK-2saVl0&hl=en&ei=R4KpTLLbA8P88Aab14zhDA&sa=X&oi=book_result&ct=result&resnum=9&sqi=2&ved=0CFEQ6AEwCA#v=onepage&q=micro%20glass%20diaphragm%20pump&f=false
The link describes some micro sized pumps with thin glass with magnetic material bonded on.
A DIY version could be cover slip glass with some steel glued on, insulated, and
a coil mounted above each one. Driving the coils can be low volts easy. The differential pump
idea means you need no valves, also easy.
"ferromagnetic nanoparticules with external permanent magnet
could perhaps be a good valve mimic."
Thanks, but it's OK to just NOT have a valve.
On 10/04/2010 09:12 AM, ByoWired wrote:
> The biggest expense is probably the detection equipment - argon
> lasers, photomultipliers, high speed electronics, optical filters,
> etc.
Ah... well then what lower performance methods could work... I think the selecting
can be done at 240 times per second with the mentioned micropumping via fairly simple
constructions of glass, and maybe even easier if molded from agarose, so now think
about the detecting inexpensively. If you use agarose, locating the detector precisely
to sense the selecting zone would need to be done with aid of a microscope every time
I think. With glass, mountings could be made.
On 10/04/2010 09:54 AM, Nathan McCorkle wrote:
> if you were just making a cell sorter, you wouldn't need a gas laser
.
.
.
... for just color detection, cheap
> solid state lasers should be fine... and if you were driving them with
> decent current control circuits (pretty easy for an electrical engi
> student) they would not be too bad for concentration measurements
> either.
Are you meaning concentration of cells/volume?
>
> the control system would probably be able to be driven by a beagle
> board, its got plenty of power for image processing and such
Is image processing the way it's done? I was imagining fluorescence
and color detection only...
If the price of cell sorters went from >$100k to $5K, how many more would buy, and
how many buy at the current prices?
John
Nathan McCorkle
surface molecules, etc...
fluorescence and color detection are anlyzed via some sort of
processing... if its a single point detector vs a ccd you might call
that processing something different
> --
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Nathan McCorkle
a cheap fluid setup first
John Griessen
> the image processing\electronics would be easy, btw... definitely need
> a cheap fluid setup first
OK, that's been discussed and ideas generated. How about
telling us what you know about detecting cells floating along quickly
in a clear 76 micron side glass channel?
On 10/04/2010 10:30 AM, Nathan McCorkle wrote:
> i mean concentration of fluorophores... to determine amount of cell
> surface molecules, etc...
Does that mean relative to the whole fluid mix, or relative to a cell's
surface?
On 10/04/2010 10:30 AM, Nathan McCorkle wrote:
> fluorescence and color detection are anlyzed via some sort of
> processing... if its a single point detector vs a ccd you might
So some of the expensive cell sorters use an image detector?
Do they just look for a patch of surface to analyze, or do they
analyze shapes? Analyzing shapes seems too slow for today's tech
to handle without neural vision hardware, and I bet they don't have that yet.
Thanks,
John Griessen
ByoWired
by in-air sorting I'm referring to the "old fashioned" method that
creates droplets in air, charges each droplet according to which
direction you want it to go and then passes those airborne droplets
between two electrodes whose E-field guides them to their desired
location (save or discard).
On Oct 4, 10:54 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> maybe instead of 10 liters of buffer, you could deposit the sorted
> start sorting again...
The principle is good but in practice it sounds like "Welcome to
Contamination City." I'm hoping for a simple, more streamlined
approach rather than a robotic tour de force.
Brian Degger
Saw a Union Biometrica's Complex Parametric Analyser and Sorter
(COPAS) machine the other day at a marine lab that is used to sort and
identify plankton.
Given that plankton are three dimensional objects, I am assuming that
there is some shape analysis going on and that the machine is trained
in this identification.
Cheers,
Brian
ByoWired
On Oct 4, 11:09 am, John Griessen <j...@industromatic.com> wrote:
> If the price of cell sorters went from >$100k to $5K, how many more would buy, and
> how many buy at the current prices?
many others are trying to produce low-cost systems for use in
developing countries. Costs are greatly reduced if lasers can be
replaced by on-chip LEDs (used to activate fluorescent probes, etc.)
and light sensors that can pick up the emitted fluorescence.
Personally, that's not what I'm aiming to do at this point. I think
all of that is very dependent on mass produced high volume microfab,
not DIY. But of course there is always the possibility of cross
pollination of ideas if DIY can find a cheaper way.
As I said earlier, for me the electronics is not a problem (I hope).
It's the DIY tiny tubing department that leaves me baffled. I know
there are concepts galore, but so far I haven't found any technique
that's been (at least somewhat/sorta) proven/very promising for DIY
useage.
Also, more generally speaking, the microfluidics approach is super
slow compared to airborne sorters. The fluid in air-borne cell sorters
typically runs through the microtubing at about 10 meters/second.
And sorting 300 cells per second for microfluidics is a whole
different ball game compared to 10000 or 50000 cells/second, which is
possible with old fashioned airborne devices.
It's too bad there's no such thing as plug-and-chug modular components
for microfluidics in general. I think I heard Corning might be
working on such a thing, but so far I haven't seen anything on the
market.
ByoWired
On Oct 4, 11:55 am, John Griessen <j...@industromatic.com> wrote:
>
> So some of the expensive cell sorters use an image detector?
> Do they just look for a patch of surface to analyze, or do they
> analyze shapes? Analyzing shapes seems too slow for today's tech
> to handle without neural vision hardware, and I bet they don't have that yet.
>
molecular probes, etc. with lasers.
There's not an image per se, though I've heard of systems that might
look at laser scatter.
With cells whizzing by the detectors at 10 meters/second, it's hard to
get the little rascals to strike a pose. You always catch them with
their eyes closed, I bet.
As for what sort of electronics to use, everybody is going to have
their preferences. And I bet that's always going to be one of the big
stumbling blocks in any DIY community. In bio, advances are going to
become more and more dependent on electronics, sensors and
automation. And with everyone doing different platforms, it's going
to get messy when newbies just want to walk onto the scene and grab
something off the shelf and start working without really having to sit
down and learn all the code, hardware, etc from scratch. And who
wants to learn 20 different platforms, know 10 different computer
languages, to run 25 different pieces of lab equipment when really all
you want to do is get down to the business of the research itself?
I'm thinking that's just going to be the nature of the beast. Man, I
don't even know what Python is. I don't even like computers to start
with, but a couple years ago I just had to bite the bullet and learn
something.
Cathal Garvey
To be Fair, almost every other mention of embedded or microprocessing is usually arduino. As far as agreement on electronics platforms goes, we're pretty successful.
That's not to say you're wrong up use your favourite system, but when recommending systems I'd usually recommend arduino, for the future's sake.
---
Twitter: @onetruecathal
Sent from my beloved Android phone.
On 4 Oct 2010 19:08, "ByoWired" <byow...@gmail.com> wrote:
On Oct 4, 11:55 am, John Griessen <j...@industromatic.com> wrote:
>
> So some of the expensive ce...
For the most part they look at fluorescence emission after activating
molecular probes, etc. with lasers.
There's not an image per se, though I've heard of systems that might
look at laser scatter.
With cells whizzing by the detectors at 10 meters/second, it's hard to
get the little rascals to strike a pose. You always catch them with
their eyes closed, I bet.
As for what sort of electronics to use, everybody is going to have
their preferences. And I bet that's always going to be one of the big
stumbling blocks in any DIY community. In bio, advances are going to
become more and more dependent on electronics, sensors and
automation. And with everyone doing different platforms, it's going
to get messy when newbies just want to walk onto the scene and grab
something off the shelf and start working without really having to sit
down and learn all the code, hardware, etc from scratch. And who
wants to learn 20 different platforms, know 10 different computer
languages, to run 25 different pieces of lab equipment when really all
you want to do is get down to the business of the research itself?
I'm thinking that's just going to be the nature of the beast. Man, I
don't even know what Python is. I don't even like computers to start
with, but a couple years ago I just had to bite the bullet and learn
something.
--
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To po...
ByoWired
On Oct 4, 6:15 pm, Cathal Garvey <cathalgar...@gmail.com> wrote:
> To be Fair, almost every other mention of embedded or microprocessing is
> usually arduino. As far as agreement on electronics platforms goes, we're
> pretty successful.
>
> That's not to say you're wrong up use your favourite system, but when
> recommending systems I'd usually recommend arduino, for the future's sake.
>
Arduino websites and Wikipedia and I haven't found any mention of
parallel processing. Maybe it's not needed for most low-tech things
like incubators, etc. Does it have built-in hardware for things like
VGA (running a computer monitor to display real time data)? I don't
have any experience with other systems, so I don't really know how to
make comparisons. Here in the US, some Radio Shack stores have
beginner kits "What's a Microcontroller?" and I found that makes a
good way to start.
John Griessen
> It's too bad there's no such thing as plug-and-chug modular components
> for microfluidics in general. I think I heard Corning might be
> working on such a thing, but
Corning has chemicals for bonding glass and promotes courses in cutting and grinding smooth,
then bonding as an assembly method for human scale glass.
I think it's as good as fusion weld in glass. It will almost certainly be good enough
for room temperature bonding glass to make microfluidic circuits.
On 10/04/2010 01:08 PM, ByoWired wrote:
>> So some of the expensive cell sorters use an image detector?
>>
> For the most part they look at fluorescence emission after activating
> molecular probes, etc. with lasers.
> There's not an image per se, though I've heard of systems that might
> look at laser scatter.
> With cells whizzing by the detectors at 10 meters/second, it's hard to
> get the little rascals .. .
Thanks, I'll look into that. You know, one way to get 5000 cells per second is
with ten parallel 500 cell per second machines. Machines that cost 1/100
as much to make as the fastest ones. And if they're small, and the user interface hides
the complexity from you, you might even buy one.
JG
Nathan McCorkle
> Hi Nathan,
>
> by in-air sorting I'm referring to the "old fashioned" method that
> creates droplets in air, charges each droplet according to which
> direction you want it to go and then passes those airborne droplets
> between two electrodes whose E-field guides them to their desired
> location (save or discard).
Ok, I was thinking it would be sideways drops, like a gun or something!
>
> On Oct 4, 10:54 am, Nathan McCorkle <nmz...@gmail.com> wrote:
>> maybe instead of 10 liters of buffer, you could deposit the sorted
>> cells into something like a cement mixer... pellet cells, suck off fluid, recirculate buffer,
>> start sorting again...
>
> The principle is good but in practice it sounds like "Welcome to
> Contamination City." I'm hoping for a simple, more streamlined
> approach rather than a robotic tour de force.
>
Could you do concentration in a microfluidic setup easily?
ByoWired
On Oct 5, 1:38 am, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> Could you do concentration in a microfluidic setup easily?
>
think of a good way to make a cheap sorting valve. :-)
I suppose there might be ways of using filters to concentrate cells,
but the smaller the filter pore size, the harder it gets to push fluid
through it, therefore you need huge amounts of surface area, and large
filters cost big bucks. Then you have to harvest the cells, which
might be scattered all over the filter material.
If you're talking about recirculating the process fluid, well, since
you've just shot the droplets through the air, you'll probably want to
cut down on possible contamination of the fluid somehow - either using
submicron filters ($$$$) or sterilizing the fluid before recirculating
it (planet Mercury-style UV exposure?)
Also, the word "easily" seems very subjective around here. I've heard
people mention things that are "easy" which I would categorize as
"okay, it's doable, but hold on to your butt."
:-)
Mark
Nathan McCorkle
> If you're talking about recirculating the process fluid, well, since
> you've just shot the droplets through the air, you'll probably want to
> cut down on possible contamination of the fluid somehow - either using
> submicron filters ($$$$) or sterilizing the fluid before recirculating
> it (planet Mercury-style UV exposure?
blood IV sets are sterile and have a drip tube... commercial cell
sorters must have some sort of chamber... glass or plastic that was
autoclavable would work..
>
> Also, the word "easily" seems very subjective around here. I've heard
> people mention things that are "easy" which I would categorize as
> "okay, it's doable, but hold on to your butt."
>
> :-)
> Mark
>
Nathan McCorkle
> On 10/04/2010 10:32 AM, Nathan McCorkle wrote:
>>
>> the image processing\electronics would be easy, btw... definitely need
>> a cheap fluid setup first
>
> OK, that's been discussed and ideas generated. How about
> telling us what you know about detecting cells floating along quickly
> in a clear 76 micron side glass channel?
>
current cell sorting machines are FACS (flourescence-activated cell
sorting), they are based off of flow cytometers because flow
cytometers are really good at detecting cells and features about them,
labelled and unlabelled. When you illuminate an object, there is
scattering, so you have a bunch of ways to measure this.
back-scattering looks at light returning in the direction of
illuminaton... it uses a half-silvered mirror, you bounce light into
the mirror and onto a flow cell, then collecting through the backside
of the mirror. side-scatter is when you collect light 90 degrees right
of illumination.
see my drawings here for rough idea:
http://nathanmccorkle.com/projects/scatter.pdf
From Beckman-Coulter:
Side Scatter (SS) The amount of light scattered at a right angle to
the direction of the laser beam. The amount of light scattered is
directly proportional to the complexity of the internal structures
and/or internal granularity within a particle.
Forward Scatter (FS) The laser light scattered at narrow angles to
the axis of the laser beam. The amount of forward scatter is directly
proportional to the size of the cell that scattered the laser light.
(from Glossary at: http://coulterflow.com/bciflow/research01.php)
> On 10/04/2010 10:30 AM, Nathan McCorkle wrote:
>> i mean concentration of fluorophores... to determine amount of cell
>> surface molecules, etc...
>
> Does that mean relative to the whole fluid mix, or relative to a cell's
> surface?
>
well thats a measure of resolution... the cells' surface would be fine
resolution, which good systems have... otherwise you would need just
enough signal to give you a pattern to select against.
resolution depends on: light source brightness, collector sensitivity
(shutter speed, % photons converted to electrons(quantum efficiency)),
collection scheme(for colors you would need to pass the light into a
prism or through/reflected off a grating... for shape detection and
colors you might just add a spectral color sensor to some other area,
like behind the cell...)
If you were just looking for color patters, I think either scatter
approach would be good, using broadband light off the sample and
(reflected off)/(passed through) a grating to a linear array detector.
This linear array CCD has a max data rate of 1MHZ (analog signals) and
3648 pixels that are 8um x 200um (w * h)... so thats 2.9184
centimeters of pixels to focus the quantized spectra. If I am correct,
you take the data rate divided by data number (1 MHZ/3648 pix = 274
frames/second).... that's pretty fast, and its a damn good sensor...
for $20 on ebay...
www.spectralproducts.com/catalog/pdf/TCD1304AP.pdf
it would require a real nice ADC, at least 10-bit if not more... if
its 10-bit (2^10 = 1024 possible intensity values per pixel) then each
will be stored in a 16-bit integer for memory alignment... so (1MHZ *
16 bits = 1.9 megabytes/second.... each frame would be 7.125
kilobytes) 3648 values populate an array (ideally) every 3.648
milliseconds ( (1 second) / (1 000 000 / 3648) ) and in that time you
have to do all processing... e.g. finding calibration peaks or edges,
finding sample peaks and identifying a pattern, and then actuating the
selection equipment.
if anyone familiar with arduino can make sense of these numbers in
terms of performance, that'd help determine what processor to use.
Texas Instruments makes the processor on the beagle board that runs at
about 600MHZ optimized for fixed point math I believe. Its got a
second core that runs linux, for handling user interfaces and
displaying/logging results. Linux could even do some graph building in
real-time. beagle board =~ $100
I analyze shapes at work using a similar system on much larger (2D)
images at 60 FPS... so doing this should be no problem. To analyze the
traditional scatter images, you would just use another processor so
each sensor would be by itself.
> On 10/04/2010 10:30 AM, Nathan McCorkle wrote:
>> fluorescence and color detection are anlyzed via some sort of
>> processing... if its a single point detector vs a ccd you might
>
> So some of the expensive cell sorters use an image detector?
> Do they just look for a patch of surface to analyze, or do they
> analyze shapes? Analyzing shapes seems too slow for today's tech
> to handle without neural vision hardware, and I bet they don't have that
> yet.
>
> Thanks,
>
> John Griessen
>
Mackenzie Cowell
It's too bad there's no such thing as plug-and-chug modular components
for microfluidics in general. I think I heard Corning might be
working on such a thing, but so far I haven't seen anything on the
market.
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Nathan McCorkle
> http://en.wikipedia.org/wiki/Magnetohydrodynamics#Engineering
> http://en.wikipedia.org/wiki/Magnetohydrodynamic_drive
> This is the concept I was talking about, by the way. In case anyone thought
> I was mad, apparently it works and has at least once been used for
> microfluidic PCR!
>
> Here's a good looking review:
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768299/
>
#Engineering section Microfluidic PCR sentence as 'citation needed'...
luck me I'm on DIYbio and found a good answer in this old thread!
Nathan McCorkle
Yes
Is this group still active?Im just starting out in DIY microfluidics and would love to learn from your experiences....Hopeful,Jack
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AKS
hosts handy and low-cost techniques useful for DIY.
Cheers!
On 9/21/12, diy...@googlegroups.com <diy...@googlegroups.com> wrote:
> =============================================================================
> Today's Topic Summary
> =============================================================================
>
> Group: diy...@googlegroups.com
> Url: http://groups.google.com/group/diybio/topics
>
> - X-ray crystallography, pics of Rosalind Franklin's setup... looks pretty
> DIY [1 Update]
> http://groups.google.com/group/diybio/t/37e3576a57f76d9e
> - Lab Equipment: baby bottle autoclave / sterilizer [4 Updates]
> http://groups.google.com/group/diybio/t/b66c070724d7050b
> - Anybody on the list located in Australia or New Zealand? [1 Update]
> http://groups.google.com/group/diybio/t/b5ef1493401627f2
> - Agrobacterium Ti Plasmid ID [6 Updates]
> http://groups.google.com/group/diybio/t/897ba9b1abf89039
> - Phospholipid Synthesis [1 Update]
> http://groups.google.com/group/diybio/t/a76b8ba57228b297
> - Phospholipid Synthesis [1 Update]
> http://groups.google.com/group/diybio/t/99615e0a4e4ac3a2
> - Is private funding still DIY? [4 Updates]
> http://groups.google.com/group/diybio/t/cacfc7c2d698f4f8
> - Long Term Bacterial Storage in -20C Freezer...glycerin stock? [3
> Updates]
> http://groups.google.com/group/diybio/t/65eb87bbef374c7a
> - DIY Microfluidic valves - Anyone know how to homebrew one? [2 Updates]
> http://groups.google.com/group/diybio/t/968a0b3ebdd95114
> - Citizen Science: Monitoring local water quality - link info for DIY
> measurement [1 Update]
> http://groups.google.com/group/diybio/t/ef1d0e0a9bf4f761
> - Looking for Chloroplast integration Vector [1 Update]
> http://groups.google.com/group/diybio/t/cc351140567a34c
>
>
> =============================================================================
> Topic: X-ray crystallography, pics of Rosalind Franklin's setup... looks
> pretty DIY
> Url: http://groups.google.com/group/diybio/t/37e3576a57f76d9e
> =============================================================================
>
> ---------- 1 of 1 ----------
> From: Gavin Scott <ga...@allegro.com>
> Date: Sep 21 03:34PM -0700
> Url: http://groups.google.com/group/diybio/msg/ce8476b509b6fc8b
>
> Of course there's a good chance that it killed her before she could receive
>
> her Nobel Prize too, so maybe multiple lessons for the DIYer there.
>
> G.
>
>
>
> =============================================================================
> Topic: Lab Equipment: baby bottle autoclave / sterilizer
> Url: http://groups.google.com/group/diybio/t/b66c070724d7050b
> =============================================================================
>
> ---------- 1 of 4 ----------
> From: Cathal Garvey <cathal...@gmail.com>
> Date: Sep 21 03:28PM +0100
> Url: http://groups.google.com/group/diybio/msg/7cfd6c2b32615659
>
> We had a thread a while back where we shared some papers on pure
> microwave sterilisation; apparently the measurable efficiency of
> sterilisation is comparable using a regular microwave vs. a pressure
> cooker when you're sterilising volumes less than ~100mls.
>
> So, this can only improve, IMO, when using a steam container like those
> bottle sterilisers. We have a plastic one we used early on for my
> daughter (against my microbiologist bugs-are-good-for-you objections
> :P), but I haven't nicked it for testing in the lab yet...
>
> On 19/09/12 00:59, Mackenzie Cowell wrote:
>
> --
> www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key: http://bit.ly/CathalGKey
>
>
> ---------- 2 of 4 ----------
> From: Mackenzie Cowell <m...@diybio.org>
> Date: Sep 21 11:49AM -0700
> Url: http://groups.google.com/group/diybio/msg/bc466516a246ab63
>
> Imagine you have a 500 mL beaker filled with used pipette tips, amp agar
> colonized with transformed k12 from small petri dishes, gels with DNA and
> gelgreen / sybrsafe... this all could be disposed of as normal waste by
> sterilization with a pressure sterilizer or an autoclave. What about just
> microwaves, or microwaves and 1 ATM steam?
>
> A good question for biosafety professionals.
>
> Mac
> ---------- 3 of 4 ----------
> From: Gavin Scott <ga...@allegro.com>
> Date: Sep 21 02:38PM -0700
> Url: http://groups.google.com/group/diybio/msg/a4b9805302ee6db7
>
> On Friday, September 21, 2012 1:50:05 PM UTC-5, Mackenzie Cowell wrote:
>> gelgreen / sybrsafe... this all could be disposed of as normal waste by
>> sterilization with a pressure sterilizer or an autoclave. What about just
>>
>> microwaves, or microwaves and 1 ATM steam?
>
>
> Well, I don't know, but I really doubt you're going to achieve anything
> close to sterilization that way. I think the best you're going to do is
> boiling the environment in the beaker. I'm not sure the microwaves are
> going to couple to individual critters with any degree of efficiency, and
> there's probably a big danger of nulls in the microwave field that would
> allow pockets of survivability.
>
> If your beaker is wet enough then it's just going to boil. If it's dry
> enough then you probably don't present enough of a load on the magnetron
> output and the high SWR fries your oven if you run it very long that way.
>
> As I said, I don't know, but it sure sounds sketchy to me.
>
> G.
>
>
> ---------- 4 of 4 ----------
> From: Matt Lawes <ma...@insysx.com>
> Date: Sep 21 05:53PM -0400
> Url: http://groups.google.com/group/diybio/msg/5dc44d490ea88ec
>
> The baby bottle sterilizer approach (wet steam) will work for the dry
> materials like pipettor tips and glassware. The agar plates though ..... not
> a chance. The microwave will definitely melt the agar in the plates with the
> e coli but I think you'll need some way to create pressure as well as
> boiling liquid. Perhaps if you put in a Pyrex dish with sealing plastic lid.
> Put everything in the Pyrex dish and a little water in the bottom to create
> steam. Heat in the microwave until boiling and keep it going for about 15 -
> 20 minutes. You can always test for ecoli culturability from the waste to
> assess sterility. Just a guess ..... but I bet you can optimize from there.
>
>>matt
>
> Sent from my Verizon Wireless 4G LTE DROID
>
>
> -----Original message-----
> From: Gavin Scott <ga...@allegro.com>
> To: "diy...@googlegroups.com" <diy...@googlegroups.com>
> Sent: Fri, Sep 21, 2012 17:38:57 EDT
> Subject: Re: [DIYbio] Lab Equipment: baby bottle autoclave / sterilizer
>
>
>
> On Friday, September 21, 2012 1:50:05 PM UTC-5, Mackenzie Cowell wrote:
> Imagine you have a 500 mL beaker filled with used pipette tips, amp agar
> colonized with transformed k12 from small petri dishes, gels with DNA and
> gelgreen / sybrsafe... this all could be disposed of as normal waste by
> sterilization with a pressure sterilizer or an autoclave. What about just
> microwaves, or microwaves and 1 ATM steam?
>
> Well, I don't know, but I really doubt you're going to achieve anything
> close to sterilization that way. I think the best you're going to do is
> boiling the environment in the beaker. I'm not sure the microwaves are going
> to couple to individual critters with any degree of efficiency, and there's
> probably a big danger of nulls in the microwave field that would allow
> pockets of survivability.
>
> If your beaker is wet enough then it's just going to boil. If it's dry
> enough then you probably don't present enough of a load on the magnetron
> output and the high SWR fries your oven if you run it very long that way.
>
> As I said, I don't know, but it sure sounds sketchy to me.
>
> G.
> --
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+un...@googlegroups.com.
> To view this discussion on the web visit
> =============================================================================
> Topic: Anybody on the list located in Australia or New Zealand?
> Url: http://groups.google.com/group/diybio/t/b5ef1493401627f2
> =============================================================================
>
> ---------- 1 of 1 ----------
> From: Mackenzie Cowell <m...@diybio.org>
> Date: Sep 21 12:09PM -0700
> Url: http://groups.google.com/group/diybio/msg/dc4d15d17aaad7c8
>
> Thanks, Meow-Ludo.
>
> Good info to have on hand.
>
> So the qubit requires mixing the sample with a proprietary dye? And it
> costs 1500? Lame.
>
> Mac
> =============================================================================
> Topic: Agrobacterium Ti Plasmid ID
> Url: http://groups.google.com/group/diybio/t/897ba9b1abf89039
> =============================================================================
>
> ---------- 1 of 6 ----------
> From: Cathal Garvey <cathal...@gmail.com>
> Date: Sep 21 02:58PM +0100
> Url: http://groups.google.com/group/diybio/msg/a3995f9e27c45fc4
>
> I have my doubts that it will lose its plasmid so easily. Wild plasmids,
> with all the necessary bits intact, are quite stable.
>
> Apparently one way of "curing" (the technical term) bacteria of stable
> plasmids is to grow them on agar containing an amount of Ethydium
> Bromide. Other DNA intercalators may do the same job. You have to grow
> them for many generations under these conditions; the precise number
> probably vari
> es by plasmid etc.
>
> Bear in mind; the technology to use Ti plasmids in Agrobacterium is,
> thanks to our horrendously stupid gene patenting systems, patented.
> That's why Cambria created alternatives using other species like
> Rhizobium, alternatives which are available under a pretty permissive
> (but I wouldn't say "open source") license/MTA:
> http://www.cambia.org/daisy/cambialabs/3187.html
>
> On 19/09/12 11:07, Mega wrote:
>
> --
> www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key: http://bit.ly/CathalGKey
>
>
> ---------- 2 of 6 ----------
> From: Mega <masters...@gmail.com>
> Date: Sep 21 08:30AM -0700
> Url: http://groups.google.com/group/diybio/msg/1116b98c5b9e4834
>
> Yeah, but the antibiotic resistance of another incompatible plasmid will
> hammer the other plasmids out? except if it carries the same resistance
> gene.
>
>
> ---------- 3 of 6 ----------
> From: Matt Lawes <ma...@insysx.com>
> Date: Sep 21 11:36AM -0400
> Url: http://groups.google.com/group/diybio/msg/f90461cab5d4bfad
>
> Yes if the origins of replication of the plasmids are incompatible. The
> problem is you now have the new plasmid in there instead. This approach
> would work with a temperature sensitive mutation in partitioning genes of
> the plasmid. Use the TS plasmid to kick out the first one at lower
> permissive temp with appropriate antibiotic selection, then up shift to the
> higher temp and grow without antibiotic.
>
>>matt
>
> Sent from my Verizon Wireless 4G LTE DROID
>
>
> -----Original message-----
> From: Mega <masters...@gmail.com>
> To: "diy...@googlegroups.com" <diy...@googlegroups.com>
> Sent: Fri, Sep 21, 2012 11:30:43 EDT
> Subject: Re: [DIYbio] Re: Agrobacterium Ti Plasmid ID
>
> Yeah, but the antibiotic resistance of another incompatible plasmid will
> hammer the other plasmids out? except if it carries the same resistance
> gene.
> --
> You received this message because you are subscribed to the Google Groups
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> To view this discussion on the web visit
> ---------- 4 of 6 ----------
> From: Cathal Garvey <cathal...@gmail.com>
> Date: Sep 21 05:22PM +0100
> Url: http://groups.google.com/group/diybio/msg/b32b8c438a113f57
>
> Ah yes, sorry! That's clever, that could work quite well. Provided you
> wait a few generations under selection to be pretty sure of plasmid
> replacement, and then cure the "unstable" antibiotic resistance plasmid
> by ending selection. Nice!
>
> On 21/09/12 16:30, Mega wrote:
>
> --
> www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key: http://bit.ly/CathalGKey
>
>
> ---------- 5 of 6 ----------
> From: Matt Lawes <ma...@insysx.com>
> Date: Sep 21 12:39PM -0400
> Url: http://groups.google.com/group/diybio/msg/c072380543681715
>
> I got skills, lol. Card carrying PhD microbiologist / bacterial
> geneticist....... more of a gentleman scientist these days so DIYBio is a
> cool ethos to me!
>>matt
>
> Sent from my Verizon Wireless 4G LTE DROID
>
>
> -----Original message-----
> From: Cathal Garvey <cathal...@gmail.com>
> To: "diy...@googlegroups.com" <diy...@googlegroups.com>
> Sent: Fri, Sep 21, 2012 12:22:53 EDT
> Subject: Re: [DIYbio] Re: Agrobacterium Ti Plasmid ID
>
> Ah yes, sorry! That's clever, that could work quite well. Provided you
> wait a few generations under selection to be pretty sure of plasmid
> replacement, and then cure the "unstable" antibiotic resistance plasmid
> by ending selection. Nice!
>
> On 21/09/12 16:30, Mega wrote:
>> Yeah, but the antibiotic resistance of another incompatible plasmid will
>> hammer the other plasmids out? except if it carries the same resistance
>> gene.
>
> --
> www.indiebiotech.com<http://www.indiebiotech.com>
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key: http://bit.ly/CathalGKey
> --
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+un...@googlegroups.com.
> From: Nathan McCorkle <nmz...@gmail.com>
> Date: Sep 21 02:38PM -0400
> Url: http://groups.google.com/group/diybio/msg/4cb3edf5a3a87b9c
>
>> I got skills, lol. Card carrying PhD microbiologist / bacterial
>> geneticist....... more of a gentleman scientist these days so DIYBio is a
>> cool ethos to me!
>
> *Like*
> --
> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics
>
>
>
> Topic: Phospholipid Synthesis
> Url: http://groups.google.com/group/diybio/t/a76b8ba57228b297
> =============================================================================
>
> ---------- 1 of 1 ----------
> From: Matt Lawes <ma...@insysx.com>
> Date: Sep 21 12:50PM -0400
> Url: http://groups.google.com/group/diybio/msg/465d1c2ade3c888e
>
> Hi James,
>
> Synthesizing phospholipids chemically is actually pretty hardcore organic
> chemistry (part of what my business Molecular Forges does).
> It's easier from the DIY side to extract the phospholipids from samples ....
> algae etc. You can also buy the phospholipids as kits which structural
> biologists and physiologists use to build synthetic membranes and bicelles.
>
> What are you trying to do .... there may be easier approaches?
>
>>matt
> Sent from my Verizon Wireless 4G LTE DROID
>
>
> -----Original message-----
> From: jcrubino <james....@gmail.com>
> To: "diy...@googlegroups.com" <diy...@googlegroups.com>
> Sent: Fri, Sep 21, 2012 11:39:39 EDT
> Subject: [DIYbio] Phospholipid Synthesis
>
> Hello,
> Are there any resources available on synthesising a phospholipids...
> possibly on synthesising films or ideally an empty membrane?
>
> Thanks
>
> James Rubino
> New, Interested, and Curious
>
>
> --
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+un...@googlegroups.com.
> To view this discussion on the web visit
> =============================================================================
> Topic: Phospholipid Synthesis
> Url: http://groups.google.com/group/diybio/t/99615e0a4e4ac3a2
> =============================================================================
>
> ---------- 1 of 1 ----------
> From: jcrubino <james....@gmail.com>
> Date: Sep 21 08:37AM -0700
> Url: http://groups.google.com/group/diybio/msg/bc9cbec82cb0ce2c
>
> Hello,
> Are there any resources available on synthesising a phospholipids...
> possibly on synthesising films or ideally an empty membrane?
>
> Thanks
>
> James Rubino
> New, Interested, and Curious
>
>
>
> =============================================================================
> Topic: Is private funding still DIY?
> Url: http://groups.google.com/group/diybio/t/cacfc7c2d698f4f8
> =============================================================================
>
> ---------- 1 of 4 ----------
> From: Pieter <pieterva...@gmail.com>
> Date: Sep 21 12:15AM -0700
> Url: http://groups.google.com/group/diybio/msg/1210fbcdf7239ceb
>
> Funny how paying for our own pizza, beer and arduino turns into "privately
> funded".
>
> On Friday, 21 September 2012 01:18:22 UTC+2, Jelmer Cnossen wrote:
>
>
> ---------- 2 of 4 ----------
> From: Michael Turner <michael.eu...@gmail.com>
> Date: Sep 21 04:32PM +0900
> Url: http://groups.google.com/group/diybio/msg/be2f4c5fae01c3e9
>
>> Funny how paying for our own pizza, beer and arduino turns into
>> "privately
>> funded".
>
> "Self-financed" can sound cheap. Sometimes you don't want to say that
> even if it IS cheap. Many crowdfunding initiatives aim at eventual
> conventional financing. It might be a while before having established
> an initial customer base through crowdfunding is considered a mark of
> unusual merit, to mainstream investors. I think it should be,
> actually.
>
> Regards,
> Michael Turner
> Project Persephone
> 1-25-33 Takadanobaba
> Shinjuku-ku Tokyo 169-0075
> (+81) 90-5203-8682
> tur...@projectpersephone.org
> http://www.projectpersephone.org/
>
> "Love does not consist in gazing at each other, but in looking outward
> together in the same direction." -- Antoine de Saint-Exupéry
>
>
>
>
>
> ---------- 3 of 4 ----------
> From: Bryan Bishop <kan...@gmail.com>
> Date: Sep 21 02:40AM -0500
> Url: http://groups.google.com/group/diybio/msg/f5aeb24cdf067ad4
>
> On Fri, Sep 21, 2012 at 2:32 AM, Michael Turner
>> "Self-financed" can sound cheap. Sometimes you don't want to say that
>> even if it IS cheap.
>
> Definitely. But in the context of venturebeat.com, it's sort of
> obvious which way a journalist is implying it. That's all. BTW, I
> wasn't trying to imply that a company couldn't sell products to a
> non-institutional market and not call that product DIY. There's
> definitely people willing to not buy ebay-cyclers.
>
> - Bryan
> http://heybryan.org/
> 1 512 203 0507
>
>
> ---------- 4 of 4 ----------
> From: Cathal Garvey <cathal...@gmail.com>
> Date: Sep 21 03:33PM +0100
> Url: http://groups.google.com/group/diybio/msg/7c626c43ca23b625
>
> Glad to hear (though I never doubted) that you guys are gonna stick with
> the hacker ethic and forego patents. :)
>
> I was debating this at PICNIC with our Infectious Diseases expert,
> saying that one of the big "sells" of something like Amplino is not only
> the good that can be done through foreign aid, but the fact that someone
> in an affected region could *make their own* if they needed to. That, to
> me, is one of the most important parts of the open-source biology movement.
>
> Congratulations on your big win! Can't wait to see where you go with it
> from here. I'm personally hoping it continues to be built into shoeboxes
> for a while longer! ;)
>
> On 21/09/12 08:15, Pieter wrote:
>
> --
> www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key: http://bit.ly/CathalGKey
>
>
>
> =============================================================================
> Topic: Long Term Bacterial Storage in -20C Freezer...glycerin stock?
> Url: http://groups.google.com/group/diybio/t/65eb87bbef374c7a
> =============================================================================
>
> ---------- 1 of 3 ----------
> From: Matt Lawes <ma...@insysx.com>
> Date: Sep 21 08:35AM -0400
> Url: http://groups.google.com/group/diybio/msg/aac8b56c5acd9747
>
> Nice protocol from Shamrock for agar stabs. Here's a bit more of a recipe:
>
> Makes enough for 100 stabs.
>
> 0.6 gm Agar
> 1 gm nutrient broth powder
> 0.8 gm sodium chloride
> 100 ml deionized water
>
> Autoclave in a 150 - 200<tel:150200> ml bottle. Dispense 1ml into 2 ml screw
> cap vials. The stab agar stock bottle can be microwaved later to melt if you
> don't need / want to aliquot all of it at once.
>
> Note: NB and LB are similar ..... straight substitute ... some LB mixes
> already include salt, in that case adjust recipe above. Salt level should be
> adjusted in general to the needs of your bacterium. Above recipe is standard
> E. coli / Salmonella mix.
>
>>matt
>
> Sent from my Verizon Wireless 4G LTE DROID
>
>
> -----Original message-----
> From: "Sebastian S. Cocioba" <scoc...@gmail.com>
> To: "diy...@googlegroups.com" <diy...@googlegroups.com>
> Sent: Thu, Sep 20, 2012 16:54:47 EDT
> Subject: Re: [DIYbio] Re: Long Term Bacterial Storage in -20C
> Freezer...glycerin stock?
>
> Thanks so much! You should post this on open wetware protocols. :)
>
> Sebastian S Cocioba
> CEO & Founder
> New York Botanics, LLC
>
> Sent via Mobile E-Mail
>
> On Sep 20, 2012, at 4:43 PM, shamrock
> <thmsb...@gmail.com<mailto:thmsb...@gmail.com>> wrote:
>
> A better strategy would be agar stabs, I've stored cultures (E. coli and S.
> cerevisiae) as stabs for six months without subculturing and my guess is you
> could go longer. The trick is slowing metabolism way way down by keeping the
> cells under anaerobic conditions and in the cold. To make a stab prepare
> sterile growth media (LB is what I typically use for E. coli) with agar and
> pipette about 1 ml into a 2 ml screw cap tube. I place the tube on an angle
> when I prepare the tubes so that the agar has a slanting surface when it
> hardens. When you inoculate stab the culture down into the agar and then let
> it grow for a day or so at 37 until you see visible growth. Tighten the cap
> and seal it with para-film if you want then store it at 4 degrees. To
> revive just stab down into the culture with a sterile loop and away you go.
>
> On Wednesday, September 19, 2012 11:09:28 PM UTC-4, Sebastian wrote:
> Has anyone had success storing bacteria for the long haul (1yr+) in standard
> freezers (-20C)? I know one makes a glycerol stock for -80C deep freezers
> but would the same be possible in -20C? Since it is warmer, what would the
> theoretical media refresh intervals look like? Monthly? Quarterly? Growth
> rate? Inhibitor additions? A bio-hacker might not have access to such a deep
> freezer but we all have freezers in our fridges. Thanks again and as always,
> happy biohacking!
> --
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To post to this group, send email to
> You received this message because you are subscribed to the Google Groups
> "DIYbio" group.
> To post to this group, send email to diy...@googlegroups.com.
> To unsubscribe from this group, send email to
> diybio+un...@googlegroups.com.
> From: Wes <wesd...@gmail.com>
> Date: Sep 21 05:25AM -0700
> Url: http://groups.google.com/group/diybio/msg/c204b49bd13723a8
>
> One trick is to use sterile 80% glycerol that has been autoclave sterilised
>
> (read: pressure cooker for 15 - 20 mins) while jar slightly open (a jar,
> ajar... get it?)
>
> Just won't sterilise at 100%: not enough water in it.
>
> Then use a 1/3 volume of the sterile glycerol to gently mix in with your
> concentrated bacterial cult stock to achieve a final conc of 20% glycerol.
>
> Oh, yeah, for the bacto stock, inoc maybe 20 mL LB or better yet, SB with
> MOPS overnight with shaking. Then ice immediately, spin for 'a bit' until a
>
> nice soggy pellet develops (too hard a spin makes too hard a pellet, too
> little just makes goo and no pellet really). Keep it cold. Resuspend gently
>
> and immediately freeze.
>
> Trick is to keep the cells as much in suspended animation with all them
> delicious metabolites ready to go internally for when you wake 'em up again
>
> later.
>
> Also, glycerol not so toxic. Can be a C source. Keeps cell walls/membrane
> flexible, high osmolarity: another important trick is to use at least final
>
> 1x LB and maybe higher salt conc to assist in partial desiccation of
> cells... stops them exploding when ice crystals form inside.
>
> Mammalian cells like to be gently cooled during the 'tricky stage' of ice
> crystal formation... ~ minus 2 to minus 20'C or so at ~1'C per minute.
> Gives crystals a chance to form outside cells, drawing moisture from inside
>
> cells, part dehydrating them and saving them from the approx. 10% increase
> in volume when fully frozen.
>
> Bacto cells don't seem too bothered by it, but all same if you're having
> difficulty, give it a try.
>
> Oh, if it's the plasmid you're really after for long term storage, then you
>
> can 'resurrect' it from dead cells by re-transforming from them into fresh
> cells. Electroporation tends to make go zappy zappy dead deady cells... but
>
> heat shocking can work, and the plasmid has been saved from oxygen by the
> dead bacto cell guts while frozen.
>
> Have fun!
>
> Wes Black
>
> On Thursday, September 20, 2012 1:09:28 PM UTC+10, Sebastian wrote:
>
>
> ---------- 3 of 3 ----------
> From: Cathal Garvey <cathal...@gmail.com>
> Date: Sep 21 03:24PM +0100
> Url: http://groups.google.com/group/diybio/msg/5d031b953a77efa0
>
> I gather they are still best kept in the fridge after being allowed to
> "take" at room temperature. Also, apparently survival is improved,
> whether freezing or just chilling, by cooling the cells to room
> temperature for a while before freezing (bacteria, not mammals, that
> is): this allows expression of protective "cold shock" proteins.
>
> Again, sorry if I can't provide the citation for that one. I can't
> recall in what context I stumbled across it, but it mostly applied to
> -80 storage. Probably equally true of -20 storage.
>
> On 20/09/12 18:04, Sebastian S. Cocioba wrote:
>
> --
> www.indiebiotech.com
> twitter.com/onetruecathal
> joindiaspora.com/u/cathalgarvey
> PGP Public Key: http://bit.ly/CathalGKey
>
>
>
> =============================================================================
> Topic: DIY Microfluidic valves - Anyone know how to homebrew one?
> Url: http://groups.google.com/group/diybio/t/968a0b3ebdd95114
> =============================================================================
>
> ---------- 1 of 2 ----------
> From: biogineer <biogin...@gmail.com>
> Date: Sep 20 08:39PM -0700
> Url: http://groups.google.com/group/diybio/msg/5479c820aa127429
> Is this group still active?
>
> Im just starting out in DIY microfluidics and would love to learn from your
>
> experiences....
>
> Hopeful,
> Jack
>
>
> From: Nathan McCorkle <nmz...@gmail.com>
> Date: Sep 21 10:05AM -0400
> Url: http://groups.google.com/group/diybio/msg/4f174b7814f57722
>
> Yes
>
>
>
> =============================================================================
> Topic: Citizen Science: Monitoring local water quality - link info for DIY
> measurement
> Url: http://groups.google.com/group/diybio/t/ef1d0e0a9bf4f761
> =============================================================================
>
> ---------- 1 of 1 ----------
> From: Jason Bobe <jaso...@gmail.com>
> Date: Sep 21 06:10AM -0700
> Url: http://groups.google.com/group/diybio/msg/b8866dff6f349fb8
>
> On Thursday, September 20, 2012 2:33:41 AM UTC-4, Jonathan Cline wrote:
>
>> Suddenly very interested in what bacto baddies are growing in the ocean I
>>
>> play in (California coast), I ran across several DIY projects on citizen
>> monitoring for bacterial counts in local rivers/beaches/etc.
>
> See also the "Klein Wasser Sensor" via the Little Devices Group at MIT:
> http://www.kleinwassersensor.com/
>
> Anna Young has many cool projects, including the "solarclave":
> http://littledevices.org/people/anna-young/
>
> Jason
>
>
>
> =============================================================================
> Topic: Looking for Chloroplast integration Vector
> Url: http://groups.google.com/group/diybio/t/cc351140567a34c
> =============================================================================
>
> ---------- 1 of 1 ----------
> From: Andreas Sturm <masters...@gmail.com>
> Date: Sep 21 01:11PM +0200
> Url: http://groups.google.com/group/diybio/msg/f741feffa2b4c0a8
>
> Definitely too much. I'll cut away some bps to make it 600 bp flanking per
> side, that will save us 400+400 bp of synthesis cost. :)
> And will work as well? Or will it give 500% more transformants, then I'll
> leave it??
>
>
>
>
>
>
>
>
>
> --
> You received this message because you are subscribed to the Google Groups
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> To post to this group, send email to diy...@googlegroups.com.
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