Re: [DIYbio] Re: DIY electroporation protocol

Is the pGLO plasmid amp resistant?  I would also try plating directly (1-2 minutes) after adding LB, if the pGLO plasmid is amp-resistant, to reduce possible satellite colonies if it works better next time.

--A

On Wed, May 9, 2012 at 5:31 PM, Nathan McCorkle <nmz787@gmail.com> wrote:

On Fri, Jan 7, 2011 at 12:31 AM, Nathan McCorkle <nmz787@gmail.com> wrote:
> So I ended up doing the CaCl2 transformation along with the class I am
> TAing (by myself this week, and I'm an undergrad!), as well I
> performed the quick and dirty electroporation too. It worked and my
> results were about 40 times as many transformants than the CaCl2 method
>
> I would have tried the piezo sparkers, but they were packed in boxes
> due to a recent lab move. Another time for sure!

So I tried piezo sparkers last week, even crappier protocol than
before though... got 2 colonies on the circumference of the agar, so
it could be contam from the incubator... subcultured them but waiting
on the results... will still need to induce with arabinose too I think

pics of the ghetto-porators
https://picasaweb.google.com/109403794341975968814/DropBox?authuser=0&authkey=Gv1sRgCIew8dDku-y-ugE&feat=directlink
Procedure (really crappy, not worth repeating in this exact way):

HB101 cells from a healthy streak plate were scraped with a sterile
toothpick, about 1cm drag of the tip.

Toothpick w/cells was beaten/swirled in 500uL sterile water in a 1.5mL
eppendorf tube

This was repeated 6 times with a fresh toothpick and a fresh
tube/water, each labelled numerically in sequence

lyophilized pGLO plasmid (bio-rad) was suspended in TE buffer to
80ng/uL, 10uL of this was added to each eppendorf with cells in it

one tube didn't have cells (control for reagent contamination), one
tube didn't get electroporated (control for anomalous growth not due
to electroporation procedure)

2 methods of electroporation (ghettoElectroPoration, or ghettoPoration)

piezo with wires soldered on, and end of wires coiled in circles
piezo with wires soldered on, and end of wires covered in conductive
aluminum foil tape (to increase surface area)

recorded which tubes i gave 1 click, 3 clicks, or 5 clicks... after
done clicking the piezo sparker, I quickly added 500uL LB broth

between tubes, I immersed the wet areas of the wires in 100% ethanol
for about 10-20 seconds, then dried with kimwipe (small paper towel)
and twirled the leads between my fingers to spin the ethanol off them

After about 2 hours of 'incubating' the benchtop, I plated 500uL per
plate (2 plates for each tube) on LB+amp

OVERALL:

I think the whole process was too dilute cell-wise.

Next I'll try LB-broth mid-log liquid culture with plasmid added
directly to it... to avoid using a centrifuge. I think I'll also try
something using the aluminum foil on a microscope slide and thin line
of cell+plasmid solution between them (to increase concentration from
a plate scraping, and adjust the spark gap.

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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