Again a question ;)
Well, for the cacl2 transformation - whatconcentration will I need???
I read 0.1 molar, 0.05molar, 0.07 molar??
What suits best??
On 6 Feb., 18:52, Mega <masterstorm...@gmail.com> wrote:
> 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
>
> > > --
> > > 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 athttp://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.
0 comments:
Post a Comment