[Open_electroporator] culture shock tests on live cells

Mitchell Altschuler mitchellalt at gmail.com
Thu Jul 13 10:41:28 UTC 2017


Just saw this message which related to my last email from this morning
...reading this now....
just need some input on next steps on testing the EPer

thanks

On Wed, Jul 12, 2017 at 4:59 PM, John Griessen <john at cibolo.com> wrote:

> On 07/11/2017 08:49 PM, Mitchell Altschuler wrote:
> > It also seems that the old EP process was changed from what was done in
> the past I assume because of the cost and safety issues ... some of those
> capacitors seemed extremely dangerous if the charge was not discharged ....
> >
> > Since I am new to this project it would be useful to know why we think
> these new parameters will still work?  Is that documented anywhere?
>
> I found plenty googling "electroporation cells" "electroporation bacteria"
> "electroporation cell wall" "electroporation voltage"
>
> Here is some of the early theory and experiment by Dr. James C. Weaver* on
> electroporation:
>
> http://onlinelibrary.wiley.com/wol1/doi/10.1002/jcb.2400510407/abstract
> http://cicbs.com/rife/Electroporation97.html
>
> His long list of references is good for finding some more papers also.
> http://www.freefullpdf.com  helped.
>
> Reading Weaver et al, you get a conclusion that if the cells are not close
> and touching by a good bit, they will
> get pores in the sides closest to either end along the lines of electron
> flow, and if they are moved around some
> you can have pores all around.  This seems natural to me from my studies
> of fields.  Pores all around gives good transfection -- it lets molecules
> go in and out of good sized holes that appear in a couple of microseconds,
> then go away, (zip up), after seconds or minutes pass.
>
> Another conclusion from this and other researchers' papers is that there
> is a threshold for making pores, after which
> they stay open and any higher volts is destructive, but repeated lower
> volts tends to move charged molecules and push and pull them through the
> pores.
>
> Beyond that it's all details.  Many of the current bragging "new tech"
> instrument sellers say they have
> made choosing the volts to transfect at automatically.  That amounts to
> zapping, then measuring the effective impedance of the whole cuvette and
> comparing it to the before condition.  When the impedance drops some, you
> infer that pores have opened, and stop
> increasing the volts then.  the culture shock will probably be able to do
> this function also and without running into any patents, since this
> phenomenon is mentioned in JCW's early papers from 1992 - 1995, which is in
> the patent expired range now.
>
> So, to give you the short answer of "why we think these new parameters
> will still work?", it's that  volts matter, and they have
> quick effect in the microseconds range, then the cells stay  "porated" for
> a while, (minutes), and that means volt level matters, and nothing in
> particular about the waveform matters.  An effective waveform from the
> theory is a peak at X volts with a top that lasts at least 10 microseconds,
> then it can fall and be jaggy or smooth, and come back up to 0.9 * X volts
> for another twenty times over 10 milliseconds falling to .5 * X volts in
> between or just stay between 0.9 * X and 0.89 * X for up to 10 milliseconds
> and be good.
>
> Either way -- smooth or jagged volts will work fine.  Square pulses or
> exponential rises and falls will work just as well.
>
> That's my reading of JCW's work.  Next to prove it with the convenient
> culture shock gear.  Much easier than experiments in 1992.
>
> --
> John Griessen
> cibolo.com  Austin TX  building lab gear for biologists
> _______________________________________________
> open_electroporator mailing list
> open_electroporator at cibolo.us
> https://cibolo.us/mailman/listinfo/open_electroporator
>



-- 
Mitchell Altschuler
925 212 2296
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