Re: [DIYbio] Re: DIY electroporation protocol

Neat, thanks! :D

"Meredith L. Patterson" <clonearmy@gmail.com> wrote:

>Brilliant! I brought this up on the NPR Weekend Edition interview I
>just
>got back from, so I'm especially glad it worked ;) (You and Cathal and
>the
>mailing list all got shout-outs.)
>
>--mlp
>On May 10, 2012 8:02 PM, "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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