Re: [DIYbio] Re: DIY electroporation protocol

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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