your own electroporator

[Laureana Stelmastchuk]

Since the topic "transformation in e coli" got many messages about how to make your own electroporator, I decided to open up it's discussion (then, people can identify the topic...)
We were talking about...:


>Mega
>You don't need any resistors or capacitors or ... anything?
>
>Just an ignitor, electrodes and a cuvette??????
>
>If so, Awsome!!!

>
>On 7 Feb., 17:49, Nathan McCorkle <nmz...@gmail.com> wrote:
> one or two sparks should be sufficient as long as the volts/cm is right.
>
>

> On Tue, Feb 7, 2012 at 8:14 AM, Mega <masterstorm...@gmail.com> wrote:
> > That'll be one of my next projects... :) An electroporator....
>
> > Using an piezoelectric lighter.... Has anyone a foto of the circuit?
> > You have to push the piezoelectric botton with your hands very often,
> > or is there a circuit that transforms the voltage??
>
> > On 3 Feb., 19:55, Laureana Stelmastchuk <laure.stelmastc...@gmail.com>
> > wrote:
> >> Well, it wont answer your main questions, but could give you some great
> >> ideas...
> >> Have you ever tried electroporation? (http://en.wikipedia.org/wiki/Electroporation#Electroporators).
> >> I think electrocompetent cells are easier to prepare than the chemical
> >> ones, and it shows better transformaton rates. I have used both *E.coli *DH10B
> >> and DH5α.
> >> However, if you don't have an electroporator... I don't know, but would be
> >> cool to try to make one.



--
Laureana Stelmastchuk Benassi Fontolan
@laurestk
http://lattes.cnpq.br/0623626644642588
http://www.tumblr.com/blog/gypsyware

[Nathan McCorkle]

Read the two posts here with info compiled by Simon Quellen Field
about how to use a piezo sparker and a potentiometer to adjust the
voltage, which for E.coli is generally 18kV/cm (or 1.8kV if the
electrodes in the cuvette are 0.1cm apart).

http://groups.google.com/group/diybio/browse_thread/thread/78979422c0983c6a/6a428075296c5339#18a3502d463e9929

I've had good results just taking exponential (mid-log) phase E.coli,
rinsing them twice with sterile water, then electroporating with GFP
in a vector. This was with a commercial electroporator, but I think
the next step is to try the same protocol but replace the spark source
with a piezo element.

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

[TILAbout]

Hey everyone, I recently acquired a gene pulser ii without a shock chamber and wanted to know if anyone can help me source one? I'm looking forward to getting into DIYBio but sometimes just have to make do with what I can get. If possible I'd like to avoid purchasing a brand new shock chamber from BioRad they cost $$$. Thank you for the help. I can't wait to get started within the year!

[Nathan McCorkle]

I made one with acrylic and a bandsaw a while ago... the electrodes
should be shielded so you can't ever touch them once its sealed. Just
use standard banana plug leads to plug into the gene pulser

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[TILAbout]

I look around town and see if I can find some acrylic sheets and glue and give it a shot. I should be able to come up with something that will do using some binding posts, high voltage banana plug cables, and maybe some small electrical contacts. Thanks for the idea.

[Nathan McCorkle]

as seen here, the cuvette electrodes are on either side at the end of
a plastic tunnel... with a plastic arm extending the same length from
the where the cuvette is actually held... so you REALLY NEVER GET NEAR
THE ELECTRODES

Also, for acrylic bonding, you can use DCM (dichloromethane) solvent,
if you can find that.

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[Nathan McCorkle]

sorry:
http://www.plant.uoguelph.ca/research/homepages/raizada/Equipment/RaizadaWeb%20Equipment%20Images/12.%20micro_pulser.jpg

[John Griessen]

On 04/08/2012 07:57 PM, Nathan McCorkle wrote:
> I made one with acrylic and a bandsaw a while ago... the electrodes
> should be shielded so you can't ever touch them once its sealed. Just
> use standard banana plug leads to plug into the gene pulser

Also, it will be best to use some thick rubber insulated HV rated wire, not the 600 Volt vinyl kind.

[TILAbout]

I've been looking around and found these images (link) of the BTX shock chamber. It doesn't look like it would be to hard to make something similar out of acrylic. Does anyone know the purpose of the funny shaped head on the slider bar? Does the cuvette somehow slip into that slot so that you can pull it out easier? Any good ideas for contacts?

[Nathan McCorkle]

That was what I as talking about. You never touch the cuvette in the tunnel with the electrodes, that's the the holder stick/arm is for. Electrodes don't matter in the tunnel as the don't touch the cell solution

On Apr 9, 2012 7:26 PM, "TILAbout" <tila...@gmail.com> wrote:

I've been looking around and found these images (link) of the BTX shock chamber. It doesn't look like it would be to hard to make something similar out of acrylic. Does anyone know the purpose of the funny shaped head on the slider bar? Does the cuvette somehow slip into that slot so that you can pull it out easier? Any good ideas for contacts?

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[TILAbout]

Ah I see I see... I didn't realize until just now that the cuvette goes in upright inside the square hole on the slide handle (remember I've never actually seen one of these in real life haha). I had pictured it lying down and being pushed in. I would imagine it wouldn't be very hard to make one at all if I can't find a source for one relatively soon. Thanks again for the heads up and the tips.

[Matthew Petersen]

I found this document that describes an electroporator to be used with an existing shock chamber, and I imagine it should not be too hard to make a shock chamber that fits the specifications of the one listed. Granted, I know nothing about the listed shock chamber, but just my two cents.

[Nathan McCorkle]

Looks good! Shock chambers can be found sterile and disposable on eBay, search electroporation cuvette

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[MattP]

Ah, so you can just use those. Yeah, I imagined the easiest way would just be that.

On Saturday, April 14, 2012 11:36:59 AM UTC-7, Nathan McCorkle wrote:

Looks good! Shock chambers can be found sterile and disposable on eBay, search electroporation cuvette

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