Re: [DIYbio] Re: DIY electroporation protocol

There's a reference floating on the list somewhere, I'll try to find it.
It involved subdermal plasmid delivery in mice using a piezoelectric
sparker, with fair results! (Don't try this at home, etc.)

On 10/05/12 23:15, Nathan McCorkle wrote:
> Mice!!?? Where?
> On May 10, 2012 6:10 PM, "Cathal Garvey" <cathalgarvey@gmail.com> wrote:
>
>> 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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>


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