Re: [DIYbio] Re: DIY electroporation protocol

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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