electroporation

Azam Rahimpour via methods%40net.bio.net (by rahimpour_a from yahoo.com)
Fri Sep 4 02:41:03 EST 2009


Hi DK
I am already looking a way for enhancing electroporation rate on mammalian cells, can you please help me with some protocols or address me to helpfull literature?
best

--- On Mon, 8/24/09, DK <dk from no.email.thankstospam.net> wrote:


From: DK <dk from no.email.thankstospam.net>
Subject: Re: low LPS plasmid prep protocol
To: methods from magpie.bio.indiana.edu
Date: Monday, August 24, 2009, 8:49 PM


In article <XrFkm.179640$3m2.43857 from newsfe06.iad>, aawara from pontiff-playground.org wrote:
>In <f0d28bfd-f57c-41d1-ade8-27aaa9b1cfe0 from 24g2000yqm.googlegroups.com>,
> Nick Theodorakis <nick.theodorakis from gmail.com> wrote:
>
>> On Aug 23, 4:37?pm, Bari Zahedi <bzah... from bccrc.ca> wrote:
>>> Does anyone have a non-kit plasmid prep protocol for cell transfection that
> will generate plasmid with low LPS levels? I will be using the plasmids ?to
> transfect immune cells. Thanks!
>>
>> Doing a double-banding in CsCl after alkaline lysis should be pretty
>> low in LPS, but I doubt that's much cheaper than using a commercial
>> kit, and you need access to an ultracentrifuge and the appropriate
>> rotor.
>
>This is how my lab does it, using a VTi90 rotor that cost an arm and a leg.
>But we get both spins done in one day -  4 hours spins at 70K.
>
>After the second spin, it takes us about 6 extractions with saturated
>butanol to get the ethidium bromide out.  We precipitate the DNA out of
>CsCl using ethanol, rather than an overnight dialysis.
>
>Typically from a 400 ml culture (in terrific broth), we recover
>about 300 - 600 micrograms of pBR322-based low copy plasmids.

Wow, so much work for so little plasmid. Just out of curiosity:
why low copy plasmids for mammalian transfections? Highly 
repetitive sequences or what? One would think that simply 
by swapping an ori you'd get your purity up ~10X.

Another thought on LPS removal: LPS has lipid and DNA 
does not. LPS must bind to resins designed for lipid removal. 
Bio-Rad, Calbiochem and Pierce sell them. (The 
polymixin-based sorbents are more selective, way more 
expensive and are designed to not bind proteins so much and 
detergent removal ones do; so for DNA purification simple 
and quite cheap detergent binding resins should do fine). 

>It is, as you say, a pain.  But on the other hand, we get excellent
>transfections of primary cells (lymphocytes in our case) using nucleofactor
>electroporations.

Nucleofector and nucleofector is a marketing device. There is 
nothing new or original in it. It's just a simple electroporator, 
pretty generic HEPES-based buffer and an alkaline peptide 
that is efficiently targeted to the nucleus. In Amaxa's case, 
it is most likely MEEDTPPIKKKRKVEDL at 25 microM final, 
a neutral charge fusion of NLS of SV40 large T-antigen and 
N-terminal sequence of VP2 protein. 

The use of DNA-binding peptides (and this one, specifically) 
to enhance transfection was reported and patented at least 
10 and 5 years before the Amaxa's patent, respectively. So 
Amaxa's patent is just a scam to circumvent existing patents, 
and the "revolutionary technology" for which absolutely no 
hint is available throught the company is nothing by a marketing 
ploy. 

Not to be an asshole, but I have to ask: Aawara, do you guys 
always do empty vector transfection controls? In more than 
one papers I saw "nucleofection" used without such control. 
How do I know the effects were not due to the magic 
bufferyou observe after transfections relate to peptide's 
presense? 

DK

P.S. I have spiked plasmid with histones from Sigma to observe 
increase of electroporation efficiency 20 years ago; 15 years ago 
the same was repeated in the lab accross the hall in Paramecium 
electroporations; neither was ever published, though).
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