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low LPS plasmid prep protocol

low LPS plasmid prep protocol - Protocols and Methods Forum

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  #1  
Old 08-23-2009, 09:37 PM
Bari Zahedi
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Default low LPS plasmid prep protocol



Does anyone have a non-kit plasmid prep protocol for cell transfection thatwill generate plasmid with low LPS levels? I will be using the plasmids to transfect immune cells. Thanks!

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  #2  
Old 08-24-2009, 05:32 PM
Nick Theodorakis
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Default low LPS plasmid prep protocol

On Aug 23, 4:37*pm, Bari Zahedi <[Only registered users see links. ]> wrote:

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.

And it's a big pain.

Nick

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  #3  
Old 08-24-2009, 11:06 PM
DK
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Default low LPS plasmid prep protocol

In article <[Only registered users see links. ].net> , Bari Zahedi <[Only registered users see links. ]> wrote:

Chromatography on something like Octyl-Sepharose should, in theory, be
very effective. LPS has got to stick to it much tighter than DNA. So it
comes down to optimizing gradient of salt and/or pH in the column
running buffer.

DK

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  #4  
Old 08-24-2009, 11:34 PM
Aawara Chowdhury
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Default low LPS plasmid prep protocol

In <[Only registered users see links. ]>,
Nick Theodorakis <[Only registered users see links. ]> wrote:


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.

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.

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  #5  
Old 08-25-2009, 12:49 AM
DK
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Default low LPS plasmid prep protocol

In article <XrFkm.179640$[Only registered users see links. ]>, [Only registered users see links. ] wrote:

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).


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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  #6  
Old 08-25-2009, 01:50 AM
Aawara Chowdhury
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Default low LPS plasmid prep protocol

In <DyGkm.116669$[Only registered users see links. ]>,
DK <[Only registered users see links. ]> wrote:


Many repeats. And all that work is worth it for the reproducibility.


All the time.

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

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  #7  
Old 08-25-2009, 02:13 AM
Nick Theodorakis
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Default low LPS plasmid prep protocol

On Aug 24, 8:50*pm, Aawara Chowdhury <[Only registered users see links. ]>
wrote:

[...]

Wow, seriously? And they didn't disclose that? You'd think people
would want to know something like that.

High copy plasmids are great when you can use them. I used to max out
the Qiagen Maxi preps (> 500 ug) from 50 ml superbroth culture using
Bluescript-derived vectors, and even more yield from a CsCl prep back
when I did them (we could often pull the band just viewing under room
lighting).


It seems in that case then one advantage of using CsCl is that one can
grab only the supercoiled form and forget the nicked version.

I thought I remembered reading that nicked forms are more prevalent
when using chloramphenicol amplification (as one might when using
pBR).

Nick

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  #8  
Old 08-25-2009, 02:20 AM
DK
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Default low LPS plasmid prep protocol

In article <mrHkm.266075$[Only registered users see links. ]>, [Only registered users see links. ] wrote:


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


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.


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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  #9  
Old 08-25-2009, 07:11 AM
Bari Zahedi
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Default low LPS plasmid prep protocol


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: [Only registered users see links. ] [[Only registered users see links. ].indiana.edu] On Behalf Of DK [[Only registered users see links. ]]
Sent: August 24, 2009 7:20 PM
To: [Only registered users see links. ]
Subject: Re: low LPS plasmid prep protocol
In article <mrHkm.266075$[Only registered users see links. ]>, [Only registered users see links. ] wrote:
Good to hear! I hope it means "every time" :-)))
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.
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
_______________________________________________
Methods mailing list
[Only registered users see links. ]
[Only registered users see links. ]
________________________________________
From: [Only registered users see links. ] [[Only registered users see links. ].indiana.edu] On Behalf Of DK [[Only registered users see links. ]]
Sent: August 24, 2009 7:20 PM
To: [Only registered users see links. ]
Subject: Re: low LPS plasmid prep protocol

In article <mrHkm.266075$[Only registered users see links. ]>, [Only registered users see links. ] wrote:


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


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.


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
_______________________________________________
Methods mailing list
[Only registered users see links. ]
[Only registered users see links. ]

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  #10  
Old 08-25-2009, 02:20 PM
Dr Engelbert Buxbaum
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Default low LPS plasmid prep protocol

Am 24.08.2009, 20:49 Uhr, schrieb DK <[Only registered users see links. ]>:



I have never worked with DNA, but should ethanol precipitation not remove
most LPS with the supernatant?

Btw, the detergent removal gels, when used properly, do not bind much
protein. Rigaud's group has done extensive work on that, for a review see

@article{Rig-98,
AUTHOR= {J.L. Rigaud},
TITLE= {Membrane proteins: functional and structural studies using
reconstituted proteoliposomes and 2-{D} crystals},
YEAR= {2002},
JOURNAL= {Braz. J. Med. Biol. Res.},
PAGES= {753-766},
VOLUME= {35},
DOI= {10.1590/S0100-879X2002000700001},
URL= {http://www.scielo.br/pdf/bjmbr/v35n7/4512.pdf},
LANGUAGE= {engl}
}
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