Re: [DIYbio] Re: DIY electroporation protocol

I should never have doubted you! :)

Thanks Nathan, awesome work and an encouraging outcome!

I wonder if you can improve efficiency with some buffer jiggery-pokery, or by altering spark-gapping..

Hmm! So, things confirmed you can electroporate with a gas lighter: E.coli, Mice. :P

Nathan McCorkle <nmz787@gmail.com> wrote:

>I did that control, I must've forgot to mention
>
>I'm gonna repeat again in next few days
>On May 10, 2012 3:27 PM, "Cathal Garvey" <cathalgarvey@gmail.com>
>wrote:
>
>> Awesome news, but you need a negative control with plasmids, cells,
>but no
>> electroporation, to be certain. Can you repeat? Lab strain E.coli can
>be
>> spontaneously transformed at a very low frequency just by adding DNA,
>> sometimes..
>>
>> Nathan McCorkle <nmz787@gmail.com> wrote:
>>
>> >Seems that one of the two colonies has pGLO! So overall the protocol
>> >works,
>> >but I'll do some more tuning to increase the efficacy
>> >
>> >Pic added to the Picassa link I posted earlier
>> >On May 9, 2012 10:09 PM, "Nathan McCorkle" <nmz787@gmail.com> wrote:
>> >
>> >> On Wed, May 9, 2012 at 9:05 PM, John Griessen
>> ><john@industromatic.com>
>> >> wrote:
>> >> > On 05/09/2012 04:31 PM, Nathan McCorkle wrote:
>> >> >>
>> >> >> I think the whole process was too dilute cell-wise.
>> >> >
>> >> >
>> >> > The 1 cm drag was starting point, then instead of streak
>reduction
>> >> > of concentration you mention fresh toothpicks. 6 times.
>> >> >
>> >> > Does a fresh toothpick have any cells on it, or sterile?
>> >> > Not understanding all of it.
>> >> >
>> >> > 6 shaking dilutions with the *same* toothpick would be a huge
>> >dilution
>> >> > factor --
>> >> > PPM I'd guess.
>> >>
>> >> Sorry, what I meant is I used a fresh, sterile toothpick for each
>1cm
>> >> drag along a lawn of E.coli, each drag got swirled in its own tube
>> >> that had 500uL sterile water
>> >>
>> >> What I meant re 'too dilute' was that I think the solution's cell
>> >> concentration needs to be higher. I could just try to scrape 6cm
>of
>> >> cell law per toothpick, which would increase the concentration of
>> >> cells 6X. My reasoning is that there just weren't enough cells to
>get
>> >> in the way of the DNA particles crashing towards the positive +
>side
>> >> of the circuit during the pulse, basically like an all
>electro-liquid
>> >> gene gun. I could also increase the concentration of the DNA, but
>the
>> >> cells are easier and cheaper to get than the plasmid.
>> >>
>> >> >
>> >> > John
>> >> >
>> >> >
>> >> > --
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>> >>
>> >>
>> >>
>> >> --
>> >> Nathan McCorkle
>> >> Rochester Institute of Technology
>> >> College of Science, Biotechnology/Bioinformatics
>> >>
>> >
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