Designing a DIY Gene Electroporator
Nathan McCorkle
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
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/