Re: [DIYbio] Re: DIY electroporation protocol

There's a paper reference floating around the list archives, I'm sure. I might have a copy in the lab too, will check when I'm in if I remember. :)

Nathan McCorkle <nmz787@gmail.com> 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
>> >> >> >
>> >> >> >
>> >> >> > --
>> >> >> > You received this message because you are subscribed to the
>> >Google
>> >> >Groups
>> >> >> > "DIYbio" group.
>> >> >> > To post to this group, send email to diybio@googlegroups.com.
>> >> >> > To unsubscribe from this group, send email to
>> >> >> > diybio+unsubscribe@googlegroups.com.
>> >> >> > For more options, visit this group at
>> >> >> > http://groups.google.com/group/diybio?hl=en.
>> >> >> >
>> >> >>
>> >> >>
>> >> >>
>> >> >> --
>> >> >> Nathan McCorkle
>> >> >> Rochester Institute of Technology
>> >> >> College of Science, Biotechnology/Bioinformatics
>> >> >>
>> >> >
>> >> >--
>> >> >You received this message because you are subscribed to the
>Google
>> >> >Groups "DIYbio" group.
>> >> >To post to this group, send email to diybio@googlegroups.com.
>> >> >To unsubscribe from this group, send email to
>> >> >diybio+unsubscribe@googlegroups.com.
>> >> >For more options, visit this group at
>> >> >http://groups.google.com/group/diybio?hl=en.
>> >>
>> >> --
>> >> Sent from K-9 Mail on Android
>> >>
>> >> --
>> >> You received this message because you are subscribed to the Google
>> >Groups
>> >> "DIYbio" group.
>> >> To post to this group, send email to diybio@googlegroups.com.
>> >> To unsubscribe from this group, send email to
>> >> diybio+unsubscribe@googlegroups.com.
>> >> For more options, visit this group at
>> >> http://groups.google.com/group/diybio?hl=en.
>> >>
>> >>
>> >
>> >--
>> >You received this message because you are subscribed to the Google
>> >Groups "DIYbio" group.
>> >To post to this group, send email to diybio@googlegroups.com.
>> >To unsubscribe from this group, send email to
>> >diybio+unsubscribe@googlegroups.com.
>> >For more options, visit this group at
>> >http://groups.google.com/group/diybio?hl=en.
>>
>> --
>> Sent from K-9 Mail on Android
>>
>> --
>> You received this message because you are subscribed to the Google
>Groups
>> "DIYbio" group.
>> To post to this group, send email to diybio@googlegroups.com.
>> To unsubscribe from this group, send email to
>> diybio+unsubscribe@googlegroups.com.
>> For more options, visit this group at
>> http://groups.google.com/group/diybio?hl=en.
>>
>>
>
>--
>You received this message because you are subscribed to the Google
>Groups "DIYbio" group.
>To post to this group, send email to diybio@googlegroups.com.
>To unsubscribe from this group, send email to
>diybio+unsubscribe@googlegroups.com.
>For more options, visit this group at
>http://groups.google.com/group/diybio?hl=en.

--
Sent from K-9 Mail on Android

--
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diybio@googlegroups.com.
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/diybio?hl=en.