Ah, ok.
I thought that the electricity can go through the cell wall anyway if
it's strong enough.
And exponential growth with thin cell walls is better for chemical
transformation.
On 5 Feb., 21:37, Nathan McCorkle <nmz...@gmail.com> wrote:
> I thought I stressed that in electroporation the cells do need to be
> mid-log/exponential phase. This is important because they're in a very
> healthy state nutrient-wise, and as Anselm pointed out their cell
> walls may be thinner (I've never heard that mentioned before, but I'm
> inclined to say it sounds reasonable)
>
> Graphite sounds like decent electrodes, only one way to see if it works!
>
>
>
>
>
>
>
>
>
> On Sun, Feb 5, 2012 at 4:10 AM, Mega <masterstorm...@gmail.com> wrote:
> > Well that sounds great...
>
> > What I have planned so far:
> > Do a calcium chloride transformation and one PEG transformation and
> > see what works best under garage conditions.
>
> > DIY electroporation also sounds great (do the cells in the
> > electroporator have to be in exponential growth?)
>
> > For the electrodes I might try graphit, from an old battery (as my
> > former chemistry teacher recommended for making electrolysis - does
> > not corrode )
>
> > On 5 Feb., 08:40, Nathan McCorkle <nmz...@gmail.com> wrote:
> >> On Sat, Feb 4, 2012 at 11:08 PM, Anselm Levskaya <levsk...@gmail.com> wrote:
> >> > Tips:
>
> >> > Ampicillin - most definitely add it to the agar, you want a uniform
> >> > concentration of the stuff. Top spreading takes forever. Just add
> >> > the amp to your agar mix after it's cooled down to merely "hot" and
> >> > not boiling.
>
> >> > Amp is decent at selecting from anywhere around 2ug/mL up to
> >> > 1000ug/mL, we typically use 50-100ug/mL in the lab. A light "dusting"
> >> > per plate by eye is generally enough. 100mg/L will work fine as a
> >> > recipe.
>
> >> > Amp is very convenient in that you don't have to wait for the
> >> > resistance gene to be expressed before adding it. Since it attacks
> >> > cell wall growth and not translation, you can just add it. With
> >> > kanamycin, chloramphenicol, etc. you have to wait an hour.
>
> >> > ---
> >> > Re competent cells: I agree that the TSS competency method is
> >> > probably the easiest to get to work in a home lab setting.
> >> > electrocompetent preps are even easier but you'd then need to
> >> > build/obtain a 2500V exponential wave shocker. (Highly recommend
> >> > electrocompetent methods if you want really high competency, i.e. if
> >> > you ever want to try a random library screen / selection.)
>
> >> Mega this is pretty good info, you should try using a piezo electric
> >> sparker, commonly found in electronic ignition household butane
> >> lighters (the long grill lighters generally have them).
>
> >> Read the two posts here with info compiled by Simon Quellen Field
> >> about how to use a piezo sparker and a potentiometer to adjust the
> >> voltage, which for E.coli is generally 18kV/cm (or 1.8kV if the
> >> electrodes in the cuvette are 0.1cm apart).
>
> >>http://groups.google.com/group/diybio/browse_thread/thread/78979422c0...
>
> >> I successfully electroporated mid-log phase (phase is important as
> >> Anselm said) E.coli with no preparation to the cells other than 2
> >> rinses with distilled and filtered water (filtered of ions such that
> >> the resistance of the water is about 18 Mega Ohms) (though distilled
> >> and sterile should be just fine).
>
> >> I did use a commercial electroporator, and commercial cuvettes (you
> >> have to be careful of the metal used for the electrodes if you make
> >> your own reactor, use gold or platinum, because they are pretty
> >> non-reactive metals if they come into solution from the spark)
>
> >> --
> >> Nathan McCorkle
> >> Rochester Institute of Technology
> >> College of Science, Biotechnology/Bioinformatics
>
> > --
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>
> --
> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics
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