Designing a DIY Gene Electroporator

[Nathan McCorkle]

So according to:
http://www.nhm.ac.uk/resources-rx/files/genepulser-76475.pdf

The Bio-Rad gene pulser can put out 2.5 kilovolts max, with (1.0,
3.0, 10, 25, or 50 µF) capacitors
"
Output voltage adjustment 50–2,500 V range (depending on the capacitor)
with 10 V adjustment precision for the high voltage
range and 2 V for the low-voltage range
(50–500 V)
"

An optional controller adds resistance, to reduce current (not volts):
"Selectable resistance, ohms, (parallel) 50, 100, 200, 300, 400, 500,
600, 700, 800, 900, 1000, infinity ohms"

An optional capacitance increaser:
"25 to 3,275 µF, in 25 µF increments"

Typical E. coli procedure:
Select a low capacitor (1.0, 3.0, 10, 25, or 50 µF)

Set the voltage, Recommended voltage is 1.8 kV for electroporation of
a typical strain of E. coli using 25–40 µl of sample
(cells/DNA/protein) in the 0.1 cm cuvette, or use 2.5 kV in 0.2 cm
cuvettes

Select resistance

Pulse the previously chilled cell sample

Immediately add recovery medium, gently mix and transfer to a culture
tube for incubation

After about an hour plate on selective medium

-----------

On Tue, Nov 8, 2011 at 6:02 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
> Why not use electroporation for transfection... I've got this book:
> http://pages.towson.edu/jsaunder/Saunders%20Publications/48.Guide%20to%20Electroporation%20and%20Electrofusion.pdf
>
> and its like the holy-grail for electroporation. Techniques and
> reviews of systems, and it even talks about building your own (the
> advantages and disadvantages mainly).
>
> "The exponentially decaying wave generator gave high rates of both
> uptake and expression; however,
> the pulse field strength working range was very narrow. Regardless of
> which wave generator is used, it is clear that the
> experimental protocol must be optimized for each cell type
> that is being examined. The optimization often involves the
> use of different electroporation chambers. The cuvette-style
> electroporation chamber (13) increases the ease and simplicity
> in the handling of cells during electroporation and has evolved
> as an industry standard"
> from:
> http://www.plantphysiol.org/content/99/2/365.full.pdf
>
> Exponential decay is what capacitors do, and I know we've got some
> Electrical Engineers active on this list... I'm no electrical expert,
> but I think I remember reading that the capacitance changed the time
> of decay, so really configurable electroporators basically just have
> lots of different sized capacitors.
>
> With some thought and dedication, I think we could build an Open Electroporator.
>
> On Mon, Nov 7, 2011 at 5:03 AM, Cathal Garvey <cathal...@gmail.com> wrote:
>> Legal impediments; you need a license to own a gun in Ireland, and AFAIK
>> that does include low-caliber air-guns.
>>
>> Which is fine in my opinion; we Irish had a storied history of shooting
>> one another, and the lack of guns in the public domain mean our police
>> force is likewise unarmed. It's a nice compromise.
>>
>> However, this does lead to my focusing on chemical or biological methods
>> of gene transfection, which is probably good really: given the choice
>> between a chemical and instrumental route, the former is usually easier
>> for a toe-dipping beginner to invest in and try out. The latter is often
>> more reproducible provided everyone's using the same equipment, but
>> that's not true of a "Make your own Gene Gun!" situation. You'd need the
>> "OpenPCR of Gene Guns". And patents would probably get in the way of
>> something awesome along those lines.
>>
>> On 07/11/11 06:41, Patrik wrote:
>>> On Nov 6, 3:13 am, Cathal Garvey <cathalgar...@gmail.com> wrote:
>>>>
>>>> OTOH, in the USA you guys can always get an airgun and modify it into a
>>>> crappy biolistics gun to shoot DNA-coated particles at your plants.
>>>> There are protocols for coating particles with DNA out there.
>>>>
>>>> It'll be expensive and difficult and it's *not* for beginners, but it's
>>>> certainly possible IMHO.
>>>
>>> Yeah, I definitely think we should put some thought into a DIY gene
>>> gun. That seems entirely feasible, and could actually circumvent a lot
>>> of genetic engineering hassles.
>>>
>>> Cathal, when you say that we could do this "in the USA" - what would
>>> be the main impediment for you guys? Gun regulation issues? Or gene
>>> gun / genetic engineering issues? Just wondering.
>>>
>>
>>
>> --
>> www.indiebiotech.com
>> twitter.com/onetruecathal
>> joindiaspora.com/u/cathalgarvey
>> PGP Public Key: http://bit.ly/CathalGKey
>>
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>>
>
>
>
> --
> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics
>

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Nathan McCorkle]

The last section here is from the book I have, it talks about homemade
equipment (the prior sections talk about commercial systems)
http://pages.towson.edu/jsaunder/Saunders%20Publications/46.Pulse%20Generators%20for%20Electrofusion%20and%20Electroporation.pdf

These and more on Alibaba:
http://www.lifescientz.com/Gene-Transformation-System.htm

Another review of different systems:
http://classic.the-scientist.com/article/display/17657/

actually a lot of good articles here with electro in the name, more
parts from the "electroporation and electrofusion" book too:
http://pages.towson.edu/jsaunder/Saunders%20Publications/

[John Griessen]

On 11/08/2011 07:41 AM, Nathan McCorkle wrote:
> The last section here is from the book I have, it talks about homemade
> equipment (the prior sections talk about commercial systems)
> http://pages.towson.edu/jsaunder/Saunders%20Publications/46.Pulse%20Generators%20for%20Electrofusion%20and%20Electroporation.pdf

Sounds easy. Meredith was interested in development once, but things changed.

One category of pulse wanted is pulses or a programmable train of pulses
that add together. Those can come from capacitance switched directly up to about 500 Volts,a
and above that voltage, it makes more sense to switch the same lower voltage caps, but feed
the current into a transformer inductor to get up to 2500 Volts. The inductively
stored energy will make it go negative voltage after the capacitance discharge,
which isn't wanted, so you can use diodes to snub
or absorb that energy. The electroporator cuvette only wants so much energy, so a current limit
is good to do, and adds to safety -- but you have to not touch the zapper or ... So this kind of thing wants
some good safety lock out physical design -- covers and such.

Besides the computer-driven switched cap pulser described above,
it might be desirable to have other signal generators to precondition what's in your cuvette,
but that can be in conflict, since 2.5kV is able to jump about 1.6mm, and will
spark across relay contacts of small, cheap relays...and kill the other signal generator.
Big clunky HV relays
are available... not much way to cost reduce them though.

I wonder what the price tag for a Scientz-2C is?

John

[Simon Quellen Field]

A silicon controlled rectifier would make a better choice than a relay.

You can even get them as optoisolators, so your Arduino can treat the

switch like an LED.

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[Simon Quellen Field]

Here's a circuit I haven't seen mentioned in this thread:

"http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CEYQFjAA&url=http%3A%2F%2Fwww.biosci.missouri.edu%2Fsmithgp%2FPhageDisplayWebsite%2Felectroporator.doc&ei=vIS5Tr23DamWiQLsidS_BA&usg=AFQjCNHbV0PgscqGzxtmFzMMFcfigiF7hw&sig2=V-qYMGYpsT5Drd5WymMBUA"

This one has some nice drawings:

"http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McCord/electroporation.htm"

Here is a PIC based microprocessor circuit that seems to use only 100 volts

(a dozen nine-volt batteries anyone?):

"http://eprints.ecs.soton.ac.uk/15013/1/53.pdf"

There's also a circuit in the Eppendorf patent:

"http://www.freepatentsonline.com/6103084.pdf"

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[Nathan McCorkle]

I think $250, but another listing says closer to $2k
http://www.alibaba.com/product-gs/228918631/Gene_electroporator.html
http://www.alibaba.com/product-gs/501806460/Gene_Electroporator.html

There's this one too, accessories look a bit strange:
http://www.alibaba.com/product-gs/226593685/Gene_electroporation.html

>
> John

[John Griessen]

On 11/08/2011 01:36 PM, Simon Quellen Field wrote:
> A silicon controlled rectifier would make a better choice than a relay.

You may have something -- I think SCRs are available for very high voltages.
The relay is only needed to protect a HF generator from the discharge generator
by disconnecting it when it is time to discharge. An SCR able to withstand 3kV
would be good for this app. Probably more expensive than a HV relay though.

JG

[Nathan McCorkle]

On Tue, Nov 8, 2011 at 2:36 PM, Simon Quellen Field <sfi...@scitoys.com> wrote:
> A silicon controlled rectifier would make a better choice than a relay.
> You can even get them as optoisolators, so your Arduino can treat the
> switch like an LED.

When I took apart a BioRad Gene Pulser II, it was loaded with
SCRs...should have taken pictures, meant to, but didn't :(

>> diybio+un...@googlegroups.com.

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[John Griessen]

On 11/08/2011 03:16 PM, Nathan McCorkle wrote:
> When I took apart a BioRad Gene Pulser II, it was loaded with
> SCRs...

Sure. They're a natural for the below 500Volt parts of the switched capacitor pulser.
And for the 2500Volt part, a transformer and some HV diodes, and maybe a HV relay
to protect a pre conditioner (optional equipment)

John

[Nathan McCorkle]

What about the issue of finding uF sized caps for 1.8-2.5kV? Is there
a way to simulate a cap's discharge? Use inductors or something?

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-Nathan

[John Griessen]

On 10/18/2013 04:01 AM, Nathan McCorkle wrote:
> What about the issue of finding uF sized caps for 1.8-2.5kV? Is there
> a way to simulate a cap's discharge? Use inductors or something?

If you mean stretch out a pulse, yes, inductance will do that, and/or
oscillate, so you have to get specific.

And yes, computer simulations are good for fine tuning power designs --
if you know what you want as a goal. MLP said she was rebooting work
on an electroporator/melaminometer the other day...

Probably, with observations from your piezo
spark generator experiments, and some more simple tests, we could skip
reverse engineering brands like Gene Pulser and such and avoid patent attacks.
There's probably no patent on basic function though, just features like
arc suppression. The high resistive DI water method seems easiest and
lowest cost parts, since it requires the least power flow. I find a newish
product uses an optional module the size of a book adding big capacitors for
long "square wave" pulses to solutions with < 1k Ohm
cuvette resistance. Square wave is a separate system to make -- put off for now.

If common practice in convenient/costly lab gear is to provide an exponential decay
from capacitor discharge of 5, 10, 25 uF, and yet, multiple pulses are just fine and OK,
why not keep costs low by using a 2 or 5 uF cap only and repeating while necessary?

One reason might be that HV makes radio interference, and it's best to make one big zap
then be quiet and say, "Who, me?" The design of this kind of thing needs
a shielding metal box to keep interference low, and when sold as a product, it needs FCC
compliance testing as an interference source. When an arc gets started, it can
generate heat and you can get a steam explosion, or just a blast of gases when in air,
so that's another reason for a solid container around the cuvette.

Leaving some of the hard parts for later, wouldn't a small capacitor fired as many times as it takes
be as good as a bigger one for the energy for a HV pulse? Does polarity matter in electroporation?
Obviously, it can't matter from the start, since orientation of bugs in a solution is random, but
after a discharge drops, what if it reverses some, giving your bugs a few wiggles of AC before dying out?
That will come naturally for a capacitor discharging into and inductive transformer to up the voltage
to 2kV.

[Nathan McCorkle]

On Fri, Oct 18, 2013 at 10:17 AM, John Griessen <jo...@industromatic.com> wrote:
> On 10/18/2013 04:01 AM, Nathan McCorkle wrote:
>>
>> What about the issue of finding uF sized caps for 1.8-2.5kV? Is there
>> a way to simulate a cap's discharge? Use inductors or something?
>
>
> If you mean stretch out a pulse, yes, inductance will do that, and/or
> oscillate, so you have to get specific.

No I meant to substitute for these seemingly hard-to-find capacitors.
Eliminating them from the design.

>
> And yes, computer simulations are good for fine tuning power designs --
> if you know what you want as a goal. MLP said she was rebooting work
> on an electroporator/melaminometer the other day...
>
> Probably, with observations from your piezo
> spark generator experiments, and some more simple tests, we could skip
> reverse engineering brands like Gene Pulser and such and avoid patent
> attacks.
> There's probably no patent on basic function though, just features like
> arc suppression. The high resistive DI water method seems easiest and
> lowest cost parts, since it requires the least power flow. I find a newish
> product uses an optional module the size of a book adding big capacitors for
> long "square wave" pulses to solutions with < 1k Ohm
> cuvette resistance. Square wave is a separate system to make -- put off for
> now.
>
> If common practice in convenient/costly lab gear is to provide an
> exponential decay
> from capacitor discharge of 5, 10, 25 uF, and yet, multiple pulses are just
> fine and OK,
> why not keep costs low by using a 2 or 5 uF cap only and repeating while
> necessary?

I read many short pulses don't get the job done, or the cells have a
much higher death rate or something.

>
> One reason might be that HV makes radio interference, and it's best to make
> one big zap
> then be quiet and say, "Who, me?" The design of this kind of thing needs
> a shielding metal box to keep interference low, and when sold as a product,
> it needs FCC
> compliance testing as an interference source. When an arc gets started, it
> can
> generate heat and you can get a steam explosion, or just a blast of gases
> when in air,
> so that's another reason for a solid container around the cuvette.
>
> Leaving some of the hard parts for later, wouldn't a small capacitor fired
> as many times as it takes
> be as good as a bigger one for the energy for a HV pulse? Does polarity
> matter in electroporation?

I believe the commercial E.coli unit I've used did single pulse per
cuvette/reaction tube. Polarity would matter in the sense that it's
going to be driving the DNA via electrophoresis... the field polarity
probably doesn't matter as much for pore formation. I bet the
difference between single long pulses and many short is that long DNA
will take some time to electrphorese from solution into the cytoplasm
through an open pore, so pulsing it will essentially reset the whole
system each time, not giving the DNA enough time to slip into and all
the way through the open membrane.

> Obviously, it can't matter from the start, since orientation of bugs in a
> solution is random, but
> after a discharge drops, what if it reverses some, giving your bugs a few
> wiggles of AC before dying out?

Probably doesn't matter for single pulses.

[Nathan McCorkle]

Ahh, I found some cheap caps ($10) that are close to brand new and not
going away any time soon as far as I can tell. Keywords: microwave
capacitor.

On Ebay I was seeing ~2kv ~1uF caps for $10, they're much larger than
that MOSFET electroporator's 'energy storage capacitor' though...
which he only lists as 3.5kV 5uF, which doesn't give me any decent
results on google.

http://www.ebay.com/itm/BiCai-1-05uF-HV-Microwave-Capacitor-CH85-21105-2100V-AC-New-/171157909970?pt=LH_DefaultDomain_0&hash=item27d9ce71d2

--
-Nathan

[Nathan McCorkle]

So with a few capacitors like this (microwave capacitor, 2kV 1uF) in
parallel (capacitance adds in parallel):
http://www.ebay.com/itm/BiCai-1-05uF-HV-Microwave-Capacitor-CH85-21105-2100V-AC-New-/171157909970?pt=LH_DefaultDomain_0&hash=item27d9ce71d2

or $1.75 each in packs of 20:
http://www.aliexpress.com/item/Free-Shipping-Imported-microwave-high-voltage-capacitor/1077977860.html

would this $7 7kV power supply be useful, could you drop the voltage
with a divider to get 1.8kV?
http://www.aliexpress.com/item/New-DC-3V-to-7KV-7000V-High-voltage-Generator-Boost-Step-up-Power-Module-free-shipping/1326494805.html

Then seems like all you'd need is an arduino, a gate driver IC, and a
MOSFET or 3.

Are these big clunky caps really the 2013 best-fit part?

--
-Nathan

[John Griessen]

On 11/03/2013 05:43 AM, Nathan McCorkle wrote:
> Are these big clunky caps really the 2013 best-fit part?

Oh, I don't know for sure. What of your guess that a few milliseconds of DC Volts are needed
to get DNA to move out of a pore? Is that anything? Do the pores close up -- how fast -- maybe
DC is unimportant?

Where is a write up of electroporation discharge where they instrumented and know the
voltage at the cuvette? The brand name I researched mentioned discharge decay curves in volts,
so I assume they did instrument theirs, but they did not mention much variety in volts, cuvette R's
energy delivered. I bet the energy delivered to a cuvette does not need to be very large.

And then there's cuvette geometry. Make a cuvette with a shorter path from side to side,
but still a usable volume and your necessary voltage just went down.
What electrode spacing do the usual suspects use? (from metal side of cuvette to other metal side.)

Before making a low cost machine, you want to know "what works", so
you can leave off features to save expense. You also figure out "what the customer wants".

From what I've read, a cap discharge is fine for high resistance solutions where the cuvette
equivalent resistance is > 1k Ohm. A special purpose machine that is no good for "square wave"
cooking of solutions with lots of salt in them with cuvette R's in the tens of Ohms can be lower cost
because it won't need as large a capacitor. The usual suspect's machine puts a selected load resistor
in parallel with the cuvette to make sure it doesn't decay too slowly and give it way more energy,
and that means about half the energy goes into the parallel load resistor, so the
caps driving ti all have to be 2X bigger. That's a feature that can be left off.

A machine with a user interface that goes through some sequences before firing off could be
programmed to measure cuvette resistance first, then balk and tell the operator "no -- I won't zap it
until you raise/lower the R of the sample."
if the cuvette R was too low or too high. Then the controller chooses a good initial voltage ,
or switches which capacitors are in the discharge circuit and asks the operator, "Zap it?".

I'm not at all interested in the DIY HV being discussed with no safety testing done,
or without thought put into safety.

I know this is about diybio, but please let's get past some of the
"quick/dirty" attitudes when discussing high voltage... and admit that when the volts go above 30 or so,
many folks just want to buy rather than make. So I'm not interested in coming up with an
instructable on how to turn a dead microwave into an electroporator. Too big a bench footprint anyway :-)

[Meredith L. Patterson]

Is there a standard reference, or much work in the literature, on pulse shape/duration/&c? I've seen several papers that essentially boiled down to "we empirically determined the most effective settings on our Bio-Rad electroporator for transforming $ORGANISM_WE'RE_INTERESTED_IN", but what is it that makes square waves so great? 

(My boyfriend the electrical engineer is the one asking this -- it'd be a lot easier for him to build a capacitor bank where the tail of each pulse trails off, and take fewer parts, but will that reduce the number of transformants?)

Cheers,

--mlp

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[Meredith L. Patterson]

Adding TQ to this discussion.

Cheers,

--mlp

[Nathan McCorkle]

On Sun, Nov 3, 2013 at 9:14 AM, Meredith L. Patterson
<clon...@gmail.com> wrote:
> Is there a standard reference, or much work in the literature, on pulse
> shape/duration/&c? I've seen several papers that essentially boiled down to
> "we empirically determined the most effective settings on our Bio-Rad
> electroporator for transforming $ORGANISM_WE'RE_INTERESTED_IN", but what is
> it that makes square waves so great?
>
> (My boyfriend the electrical engineer is the one asking this -- it'd be a
> lot easier for him to build a capacitor bank where the tail of each pulse
> trails off, and take fewer parts, but will that reduce the number of
> transformants?)
>
> Cheers,
> --mlp

This isn't specific for DNA transformation, it's talking about
chemotherapy molecules too, but it looks pretty similar to the other
stuff I've seen (and it does mention DNA quite a bit):
http://lbk.fe.uni-lj.si/pdfs/beme2004.pdf

otherwise there's this book (I've got it in paperback, I'll leaf
through it later or tomorrow):
http://www.sciencedirect.com/science/book/9780080917276

this looks OK, not much on actual devices:
http://www.physics.uoguelph.ca/~dutcher/download/handbook%20of%20biological%20physics/18.pdf

Looks like bipolar pulses can give higher transformation at lower
amplitude (voltage). One thing mentioned Electrofusion can be started
by a long-ish DC pulse, then a few AC pulses... or a DC pulse followed
by AC dielectrophoresis (the electrodes in dielectrophoresis are
covered in a dielectric insulator). Not sure how much lower AC pulses
can be than DC though...


this is quick and cheap, but some key points to refresh:
http://www.harvardapparatus.com/hapdfs/HAI_DOCCAT_3/W3_9_356.pdf


I'm sure there are more, I don't have better articles handy but there
should be some more in the DIYbio archive. See the attached paper as
well.

I can't work on this anymore today, but I'll search my papers this
week for sure.

[Nathan McCorkle]

Hmm, it seems that there aren't many people participating in general
lately on DIYbio, so maybe the traffic is good...

I guess posting more succinct updates would be good, but again, I
don't think there's much traffic at all on DIYbio, so why switch?

On Sun, Nov 3, 2013 at 4:10 PM, John Griessen <jo...@industromatic.com> wrote:
> On 11/03/2013 04:34 PM, Nathan McCorkle wrote:
>>
>> I really hate the idea of splintering
>> information that should be kept together.
>
>
> But it's a blizzard of repetitive trash from the viewpoint of most list
> memers.
>
> Why not do some editing before posting it occasionally?
>
> The diybio members are seldom interested in engineering details and if they
> are,
> summaries will attract them to the main development discussion.
>
> I elect you to cut/paste/edit/summarize and FWD: a la Bryan Bishop, to
> diybio@...
>
> John
>
> PS attached here's the flip side of that old fashioned RCA cap I put on the
> first post to the new list.
> You can find the 1st in the archives.



--
-Nathan

[Meredith L. Patterson]

So that I can look shit up later using the list name as a filter. No offense to DIYbio, but it's a much broader range of topics and electroporators get brought up in conversation often enough that it creates a lot of noise when trying to locate a particular message.

Cheers,

--mlp

--
-Nathan
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[Nathan McCorkle]

I have 42 references under diybio for elecroporation over last 4 years. That's not many to me, but hey I'll crosspost so it helps everyone.

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[Cathal Garvey]

In response to "is there a standard wave?", I'll just jump in and say:
though I've barely ever worked with electroporation, and know not what
I say, I used to work with those who did. And their impression, from
the inside, was that no: there is no standard waveform that will give
useful, if suboptimal, results across the board.

Waveform varied pretty widely even between cell types extracted from
the same species, and some strains were just too 'ard to EP without
serious work to optimise, whereas others would accept DNA if you even
threatened to buzz them a little.

That said, some thoughts:
A) EP would never have taken off if there weren't a wide margin for
error, because without knowing in advance the waveforms for this or
that species, no successes would have been likely. So some lassitude is
likely among species that are worth turning to EP to transform, to
begin with, whereas with those that don't respond without careful
optimisation, perhaps it's better to either await empirical results and
custom-build an EPorator, or turn to another method of transformation.

B) Piezos and other "dirty" electroporators might suggest that a
chaotic or noisy source offers the best route to general-purpose
transformation. That is:
- Species tend to be picky about response to particular waveforms
- Too much electricity is the chief killer when EPorating
- Efficiency at the correct waveform is *probably* scalar with
duration, implying that the most important bit is getting *any* time
at the right frequency, with everything else being a bonus.
So, perhaps rather than seeking a one-true-waveform, it might be worth
investigating a waveform that scales from one frequency to another,
covering everything in between, within the timeframe that delivers a
generally-accepted-as-OK current load on the cell suspension. So if the
cell responds to any of the intermediate frequencies or waveforms,
it'll get at least a few instants of it?

Just some thoughts, poorly grounded in experience. :)

Cathal

On Sun, 3 Nov 2013 12:15:27 -0500

"Meredith L. Patterson" <clon...@gmail.com> wrote:

[Nathan McCorkle]

Maybe we can learn from nature:
Lightning‐triggered electroporation and electrofusion as possible
contributors to natural HGT among prokaryotes
http://arxiv.org/ftp/arxiv/papers/1212/1212.5436.pdf

--
-Nathan

[Mega [Andreas Stuermer]]

I have to re-read it but I remember electrospray transfection. Very high efficiency.

[Jonathan Cline]

On Tuesday, November 5, 2013 8:36:04 AM UTC-8, Cathal Garvey wrote:

In response to "is there a standard wave?",

 

no: there is no standard waveform

That said, some thoughts:
A) EP would never have taken off if there weren't a wide margin for
error

B) 

chaotic or noisy source offers the best route to general-purpose
transformation.

C) Papers are written and experiments conducted with the equipment on-hand at the time, rarely built from scratch to test a theory or a model.   Results reflect that.  So if Joe Postdoc had an HP Signal Generator which supported three types of waves (sine, triangle, pulse) then those are the tests done (or more likely, only one of those settings is used, based on lab superstition).  Even voltages in some popular papers are based on what the equipment happened to be set to, during knob-fiddling, rather than exhaustive characterization of that protocol variable.   Remember we're talking about biologists here -- papers don't measure yield, and rarely test models.   So basically, voltage, power, wave shape, etc, has not been characterized yet.   Success of noisy sources would seem imply this too.  Throw a bunch of EMF at bio, some levels succeed.   Also characterization is a painful process if each iteration takes 5 days to complete and also includes a bunch of manual steps with potential for human error at each one.    

Build my voltage step-up circuit,  http://88proof.com/synthetic_biology/blog/archives/303

control it with my high-voltage digital control board, http://88proof.com/synthetic_biology/blog/archives/338

and zap something.

(Note I didn't say "someone".) 

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