diy electroporation so far?

[Jeswin]

Hi all,

What's become of the diy electroporation using the piezo starters? How exactly would one go about this? What's the process from beginning to end? Would I culture some cells like the NEB competent cells to use for electroporation?

I'd like to give it a try if it's not too difficult.

Thanks

[Nathan McCorkle]

From what I could tell, the pulse might have been too fast. I think
you'd need a capacitor or inductor, but not sure how much of you'd
need. Small high-voltage caps are cheap, but the ones in all the
academic publications for electroporators were all very large, the
closest I could find from consumer-land was a microwave oven capacitor
(~$10 on ebay). For an inductor, one might be able to get away with a
long piece of wire, but I don't know what thickness and/or how long.

The pulse also could have been oscillating too much, which would add a
diode to the requirements (there are also microwave oven diodes, but
likely that is overkill since the pulse is so short compared to the
1500W a lot of home microwaves use). There's a good amount of
literature on oscillating fields for electroporation, so that's why I
get this feeling.

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[John Griessen]

On 07/30/2014 12:49 PM, Jeswin wrote:
> What's become of the diy electroporation using the piezo starters? How exactly would one go about this?

I've been talking with Nathan and some others about electroporation and it seems
to range far and wide. The recipes you find usually quote using name brand equipment
with specific settings that are not in basic units of time, volts and impedance,
but a few things come out of it: 2kV is enough. Time at 2kV or falling exponentially
from it can be as brief as a millisecond and get results. Resistance of a cuvette
is about 40 kOhms in a 1mm between electrodes x 10mm x10mm cuvette.
for cultured cells in broth without adding extra salt.

It seems valid to get results
with several short pulses rather than needing long pulses.

So, here's a method for very roughly calibrating your piezo sparker:

.25 inches of spark in air is about 10k Volts at sea level. Try to find out
How long a spark your piezo zapper makes in air. If it is .4 inches, then
the Volts are: 16 kV so, to get 2kV divide by 8. For a cuvette resistance of
40k Ohms, it can be the 1:8 ratio with other resistors in series = 280 k Ohms total,
all of them rated for voltage they will carry. You could not do it with little chip resistors
that measure 2mm across for example. A way that would work OK would be 1/4 Watt resistors
of 100k, 100k, 80k Ohms where each one is about 7 mm long. Such low wattage resistors would
need short pulses only, or get hot -- higher wattage is better, but more work to find and buy.
Ordering from Digikey with a prepaid check is a good way -- they will send those orders
for zero shipping charge.

After that, just vary the number of sparks. Also you could add salinity to go below 40k Ohms
for a 1mm spacing cuvette resistance, which
would pull the volts down and spread the current through the dissolved salt as well as
the cells suspended. Both of those effects would be less volts at a any one cell,
(in case the zap is too strong -- killing them).

[Simon Quellen Field]

The piezoelectric igniters I sell on my website deliver 18 kilovolts.

I routinely power LEDs with them, so If you need to produce DC, just put an LED in series with the load.

You get a nice flash from the LED each time you click, so you get some feedback that the device is working. Nothing bad happens to the LED -- the current is so low that nothing heats up, and the voltage drops to what the LED can easily handle.

To reduce the voltage, simply put your loads in series. Assuming you want to stick with standard cuvettes, dropping the voltage by a factor of 9 simply means putting eight dummy cuvettes in series with the one that has your critters in it. Or zap 9 cuvettes full of critters at the same time.

You can use cheap off-the-shelf potentiometers as voltage dividers to dial in any voltage you like, from 18 kV down to a hundred volts or so. Again, the current is so low that nothing heats up or gets damaged, so any cheap Radio Shack potentiometer will do fine.

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[Jonathan Cline]

On Wednesday, July 30, 2014 10:50:09 AM UTC-7, phillyj wrote:

Hi all,

What's become of the diy electroporation using the piezo starters? How exactly would one go about this? What's the process from beginning to end? Would I culture some cells like the NEB competent cells to use for electroporation?

Put some culture in a cuvette, add vector, add a salt buffer, zap it with an electric field, incubate, see what grows.  I think that's the overview.

Instead of piezo starter, I am eager to have someone look at my HV power supply which can get up to 1800 VDC open circuit.  (A cuvette is an open circuit.)   The diy power supply plans are in the latest issue of BioCoder just out.

You can modify the voltage needed by changing the cuvette size (also known as, the gap creating the electromotive force).  For example many protocols state use of 2.5kV with a 2mm cuvette.  Well, you could use or make a 0.5 mm cuvette (microfludics or standard) and use a lower voltage.  You could flatten a coffee stirrer/drinking straw to a desired width, measured by micrometer.

I guess a piezo starter might work but I would think it would be rather imprecise.  Nature is imprecise too.  But you want to ensure a good result and/or a good yield.


## Jonathan Cline
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[John Griessen]

On 07/31/2014 12:45 AM, Jonathan Cline wrote:
> I am eager to have someone look at my HV power supply which can get up to 1800 VDC open circuit.

A voltage tripler like that seems fine for getting volts, and probably enough power for electrophoresis.

I'd like to design more safety interlocks before offering it as a kit instead of just a published how-to
with a "this can be deadly" disclaimer. For electroporation I am interested in using small inductor
or transformer based HV pulsers as found in laser printer supplies, since then all the HV would be in
a small box with just 24 V DC coming in from a UL listed wall wart. Smaller parts count also,
if you don't count the microcontroller. Everything wants a microcontroller these days, especially
power supplies.

[Nathan McCorkle]

On Jul 30, 2014 10:45 PM, "Jonathan Cline" <jnc...@gmail.com> wrote:

> I guess a piezo starter might work but I would think it would be rather imprecise.  Nature is imprecise too.  But you want to ensure a good result and/or a good yield.

Months ago I got no/little response when I posted a paper theorizing lightning strikes as potential for horizontal gene transfer (i.e. electroporation)

[Jonathan Cline]

Was that an earth shattering idea?   (No pun intended.)

[SC]

I've always preferred chemical transformation over electro.  No fancy-shmancy equipment needed, and it seems to work OK. 

[Nathan McCorkle]

Haha I've always preferred electroporation over chemical... No fancy buffers to worry about, just electricity!

On Aug 4, 2014 10:38 AM, "'SC' via DIYbio" <diy...@googlegroups.com> wrote:

I've always preferred chemical transformation over electro.  No fancy-shmancy equipment needed, and it seems to work OK. 

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[Mega [Andreas Stuermer]]

You still need a buffer with electrocompetent cells? 

I mean breeding E coli in LB and adding voltage and DNA won't do the job? You'll need defined conductivity, won'tt you?

[John Griessen]

On 08/06/2014 10:31 AM, Mega [Andreas Stuermer] wrote:
> breeding E coli in LB and adding voltage and DNA won't do the job? You'll need defined conductivity, won'tt you?

Nathan M. said he got good results without lowering the resistivity with NaCl.
Zapped at 40k Ohms per 1mm x 10mm x10mm cuvette with electrodes on the 10mm sides.

[Jonathan Cline]

It works OK on humans too... supposedly.

[Nathan McCorkle]

Nope I've had great luck compared to CaCl (40x better) with just water rinsed overnight cell culture:
https://groups.google.com/forum/m/#!topic/diybio/JcKyNIQGLIM

On Aug 6, 2014 8:31 AM, "Mega [Andreas Stuermer]" <masters...@gmail.com> wrote:

You still need a buffer with electrocompetent cells? 

I mean breeding E coli in LB and adding voltage and DNA won't do the job? You'll need defined conductivity, won'tt you?

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