On Sat, Mar 10, 2012 at 12:25 PM, Jeswin <phillyj101@gmail.com> wrote:
> On Sat, Mar 10, 2012 at 9:00 AM, Nathan McCorkle <nmz787@gmail.com> wrote:
>> Mega,
>> you should just try to add E.coli from a plate to sterile water in a
>> microtube. Add your DNA, then place the electrodes from a piezo
>> sparker in and spark 1-3 times (or have 3 tubes, and try 1 spark on
>> tube #1, 2 sparks on tube #2, 3 sparks on tube #3). Then add some LB,
>> and incubate for 1 hr... then plate that on LB + amp plates.
>
> What kind of E coli is this? Non-competent E coli? Where do you get
> that (I'm not sure what to search for)?
Jm109 or DH5-alpha or K12... whatever you have on hand.... i did this
once (with a commercial electroporator) and it worked wayyyy better
than CaCl2
just make sure its in exponential phase growth, that's all
I rinsed my e. coli 2 or 3 times with pure water before adding DNA and
electroporating, but Mega says he doesn't have a centrifuge... and if
he's growing on agar it is probably OK since there isn't much excess
water to begin (rinsing removes the extra salt, to reduce chance of
visibly sparking, which is too much current flowing and causes a lot
of heat and cell damage)
>
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
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