On Sat, Feb 4, 2012 at 11:08 PM, Anselm Levskaya <levskaya@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).
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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