[DIYbio] Re: DIY electroporation protocol

Again today, no cells... I'm wondering if the Aluminum caused acute
toxicity (probably not according to a paper I found), if I need higher
field strength (non-log phase cells have thicker membranes), maybe the
pulse time is too short, maybe the field isn't being setup correctly
(I can try with a commercial cuvette).

"E. coli cells in the logarithmic phase were more sensitive to PEF
[Pulsed Electric Field] treatment when compared to cells in the
stationary and lag phases (Pothakamury and others 1996)."

(supposedly from)
U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
June 2, 2000

Kinetics of Microbial Inactivation for Alternative Food Processing Technologies
Pulsed Electric Fields

http://altered-states.net/barry/rife/pulsedelectricflds.htm



High efficiency electrotransfection with aluminum electrodes using
microsecond controlled pulses
U. Friedricha, N. Stachowiczb, A. Simmc, G. Fuhrd, K. Lucase, U. Zimmermann
Bioelectrochemistry and Bioenergetics
Volume 47, Issue 1, November 1998, Pages 103–111

http://www.sciencedirect.com/science/article/pii/S0302459898001639


On Sun, May 13, 2012 at 7:22 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
> 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



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

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