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low LPS plasmid prep protocol

low LPS plasmid prep protocol - Protocols and Methods Forum

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  #11  
Old 08-25-2009, 02:52 PM
Nick Theodorakis
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Default low LPS plasmid prep protocol

On Aug 25, 9:20*am, "Dr Engelbert Buxbaum"
<[Only registered users see links. ]> wrote:

It does not. See: <http://preview.tinyurl.com/mnvkte> for example:


1.3.3.1 Rapid Isolation Micro Method for LPS (20)

1. Centrifuge a bacterial suspension (108 colony-forming units/mL in 2
mL PBS) in a 15-mL tube at 10,000×g for 5 min. Wash the pelletonce in
PBS (pH 7.2) containing 0.15 mM CaCl2 and 0.5 mM MgCl2. Resuspend
washed cells in 300µL of water and transfer to a 1-dram vial
containing a stir bar.

2. Add an equal volume of hot (65–70°C) 90% phenol. Stir the mixture
vigorously at 65–70° for 15 min. Chill suspension on ice, transfer to
a 1.5-mL polypropylene tube, and centrifuge at 8500×g for 15 min.

3. Transfer supernate to a 15-mL conical centrifuge tube. Re-extract
phenol phase with 300 µL of distilled water. Pool the aqueous phases.

4. Add sodium acetate to 0.5 M final concentration. Add 10 volume of
95% ethanol and place sample at –20°C overnight in order to
precipitate the LPS. Centrifuge precipitate at 2000×g at 4 C for 10
min. Discard supernatant.


Nick

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  #12  
Old 08-25-2009, 07:49 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:

In days before molecular biology kits, all electroporation experiments
I did used PEG precipitation after alkaline lysis. Those preps are still
very dirty! Dirtier than a regular minipreps and much dirtier than
anion-exchange purified DNA (e.g., MaxiPrep).


It wasn't a huge effect but then we never optimized anything there.
I'd even expect a very steep *decline* in efficiency above certain
threshold of histones concentration. That is because they will very
effectively shield DNA charges making it much less electrophoretically
mobile. And electrotransfection is membrane electroporation followed
by nucleic acid electrophoresis.


Here is slightly updated version of my ~ 14 years old post:

[Only registered users see links. ].
methds-reagnts/browse_thread/thread/5d5bdfec3a337482/9d08145f49fdc6b1?
hl=en&q=%2BDK+%2Belectroporation#9d08145f49fdc6b1

DK
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  #13  
Old 08-26-2009, 01:37 AM
Aawara Chowdhury
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Default low LPS plasmid prep protocol

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


Primary human B-cells that are stimulated with pokeweed mitogen. With
an Amaxa, we can get about 20% - 30% transfection, with no detectable
apoptosis.

We've been unable to transfect them using any other method.

AC
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