[DIYbio] Re: DIY electroporation protocol

I forgot to mention that between electroporations I flamed and wiped
the slide off with alcohol and a paper towel, then redrew with wax,
and flamed one last time before re-using.

There were no cells today, they're still incubating so I'll give them
another day or two.

On Sun, May 13, 2012 at 1:32 AM, Nathan McCorkle <nmz787@gmail.com> wrote:
> On May 9, 2012 5:31 PM, "Nathan McCorkle" <nmz787@gmail.com> wrote:
>> 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.
>>
>
> Ok, I added pics of the latest electroporators here:
> https://picasaweb.google.com/109403794341975968814/DropBox?authuser=0&authkey=Gv1sRgCIew8dDku-y-ugE&feat=directlink
>
>
> Electroporators:
> Aluminum adhesive-backed foil tape for electrodes
> ($7.58, Home Depot, Nashua Tape 322 1-57/64 in. x 150 ft,
> http://www.homedepot.com/h_d1/N-5yc1v/R-100030120/h_d2/ProductDisplay?catalogId=10053&langId=-1&keyword=foil&storeId=10051)
>
> Glass microscope slides were cleaned by dunking in 70-100% ethanol,
> gripping the wet slide with a microscope slide holder (springy wide
> pliers) and passing it through a bunsen burner to sterilize and dry.
> (CAUTION alcohol on slide will catch on fire)
>
> I made one and my friend Chris made one. I used two pieces of foil
> with the factory cut edges facing each other with a 1cm gap, with the
> piezo wires wrapped around the end of the glass slide, being covered
> by the foil. My friend tried using a single piece first, then cutting
> and peeling out a channel, but there was significant adhesive residue
> that would be hard to clean off.
>
> The only thing he did different was, instead of a 1cm gap, he used
> 2.1cm gap, and he taped his wires on instead of wrapping them under
> the main foil layer.
>
> We flamed the aluminum covered slides in the bunsen burner, then while
> warm drew two lines perpendicular to the aluminum electrode edges. We
> tried using Sharpie, but a wax pencil (or crayon) worked a lot better.
> My lines were about 0.2cm apart, my friends were 0.3cm apart.
>
>
> Protocol:
> Make overnight cultures of HB101 from 9pm to 5pm (20 hours), a shaking
> tryptic soy (TS) broth tube and a streaked MacConkey agar petri dish
> (LB agar would be fine too).
>
> Add 15uL sterile water to a sterile 1.5mL tube, scrape petrified dish
> about 1cm with sterile stick, twirl stick in 15uL of water to suspend
> the cells. Add to the now ~15uL of E. coli water 5uL pGLO plasmid
> (80ng/uL). Pipette this solution (~20uL total) onto the prepared
> electroporator capillary, moving the tip back and forth from one
> terminal to the other, to ensure the path between the electrodes is
> completely wet.
>
> Add 100uL sterile LB broth to sterile 1.5mL tube.
>
> Spark electroporator 3 times, with about 4 seconds between each pulse.
> Take up the liquid from the electroporator, and dispense into the tube
> containing 100uL sterile LB broth.
>
>
> My friend used his device (2.1cm gap) with a bit different protocol.
> He mixed 45uL sterile LB broth, 45uL overnight culture, and 10uL pGLO
> plasmid (80ng/uL), sparked 3 times, then transferred the liquid from
> the electroporator to a tube containing 20uL sterile LB broth.
>
> CONTROLS were prepared, one of 120uL overnight culture (to test the
> ampicillin in the plates), and one of 100uL LB + 15uL overnight
> culture + 5uL pGLO plasmid.
>
> We incubated them in a shaker for 60-75 minutes at 37 C (except the
> pure overnight 120uL subculture), then plated all 120uL from each tube
> onto separate LB+ampicillin agar plates using sterile cell spreaders,
> then incubated at 37 C upside down.
>
>
>
> They're incubating now, I'll update in a day or two!
> -Nathan



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

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