low LPS plasmid prep protocol

Bari Zahedi via methods%40net.bio.net (by bzahedi from bccrc.ca)
Tue Aug 25 02:11:29 EST 2009


Thanks all for your answers. First, appologies, i just sent an email asking Nick something i wanted to ask aawara. 
I have some follow up questions for you guys. 
First, i have too many constructs and too many transfections to be able to do CsCl plasmid purification. Any of you tried PEG ppt'n after alkali lysis and used the DNA for electroporation or nucleofection? We are not set up for CsCl purifications but used to routinely do PEG ppt'ns in the lab for sequencing back in the day.
Next, aawara, do you ever transfect GFP-tagged constructs into primary lymphocytes?If so, what was the transfection efficiency- ie % +ve cells? i would like to transfect primary murine splenic B cells using nucleofection.
DK, i'm very intrigued with your suggestion to increase electroporation efficiency with histones. The only reason i am trying nucleofection is because i get really low transfection of my cell line. I mostly first, am interested in increasing my electroporation efficiency, second, in expressing various constructs transiently in various lymphocyte cell lines. Any advice would be appreciated on how to increase my electroporation efficiency.
Thanks all!
"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)."

________________________________________
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] On Behalf Of DK [dk from no.email.thankstospam.net]
Sent: August 24, 2009 7:20 PM
To: methods from magpie.bio.indiana.edu
Subject: Re: low LPS plasmid prep protocol
In article <mrHkm.266075$E61.166305 from newsfe09.iad>, aawara from pontiff-playground.org wrote:
>All the time
Good to hear! I hope it means "every time" :-)))
>I didn't know that all nucleofactor had was a peptide - that's
>worth trying.
For a 100% reliable identification, give a small sample to
a reliable mass-spec facility, asking to look for peptides. That
it is a peptide I have no doubt. The only question is which one
of the listed is being used.
>Many years ago I was told that some of the nucleofactor
>buffers contain a low level of wortmannin.  The idea being that
>transfected DNA contains nicks etc. that activate ATM - possibly
>resulting in apoptosis of transfected cells.  The low level of
>wortmannin apparently attenuates ATM activation, while not completely
>blocking repair - permitting transfected cells time to repair DNA
>and survive.
Doubt it very much. Certainly the patent application
(US 2008/0145938) makes no mention of it.  And certainly no
evidence was ever presented that it is viability enhancement
that is a key to the claimed black magic of nucleofection.
My reading of the way the patent is written is that it mostly
has this bogus "high buffer"/"maybe, just maybe high salt"
thing (which is pretty generic anywau and is super unlikely
to be crucial) to silently slip in peptides, which is absolutely
a rip off of others' ideas.
DK
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________________________________________
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] On Behalf Of DK [dk from no.email.thankstospam.net]
Sent: August 24, 2009 7:20 PM
To: methods from magpie.bio.indiana.edu
Subject: Re: low LPS plasmid prep protocol

In article <mrHkm.266075$E61.166305 from newsfe09.iad>, aawara from pontiff-playground.org wrote:

>All the time

Good to hear! I hope it means "every time" :-)))

>I didn't know that all nucleofactor had was a peptide - that's
>worth trying.

For a 100% reliable identification, give a small sample to
a reliable mass-spec facility, asking to look for peptides. That
it is a peptide I have no doubt. The only question is which one
of the listed is being used.

>Many years ago I was told that some of the nucleofactor
>buffers contain a low level of wortmannin.  The idea being that
>transfected DNA contains nicks etc. that activate ATM - possibly
>resulting in apoptosis of transfected cells.  The low level of
>wortmannin apparently attenuates ATM activation, while not completely
>blocking repair - permitting transfected cells time to repair DNA
>and survive.

Doubt it very much. Certainly the patent application
(US 2008/0145938) makes no mention of it.  And certainly no
evidence was ever presented that it is viability enhancement
that is a key to the claimed black magic of nucleofection.

My reading of the way the patent is written is that it mostly
has this bogus "high buffer"/"maybe, just maybe high salt"
thing (which is pretty generic anywau and is super unlikely
to be crucial) to silently slip in peptides, which is absolutely
a rip off of others' ideas.

DK
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