Best E.coli strain for preparation of DNA for transient transfection.

masami yoshimura masami.yoshimura at UCHSC.edu
Tue Nov 7 15:48:51 EST 2000


Hi Max,

I have been using XL1-blue for so many years for DNA prep for 293
transfection with no problem, although my method for plasmid prep is
slightly different from yours.  I use boiling instead of alkali and 2.5 M
LiCl and PEG for DNA precipitation before CsCl eq. After EtBr removal I
precipitate DNA with isopropanol (off course you need some dilution of
CsCl).  If you need more info let me know.

Masami


T. Max M. Soegaard <tmms at mbio.aau.dk> wrote in message
news:3A07FA49.9A215D1E at mbio.aau.dk...
> Hi all.
> I have the problem that transient transfection of 293 cells by CaPO4
> precipitation now seems to kill my cells. Same construct new prep. They
> did'nt use to die like that. They seem to start dying within the first
> 16 hours after application of the transfection mix before the construct
> has a chance to express anything.
>  - Makes me think: "LPS contamination".
>
> The old prep was made in DH5alpha the new one is from XL-1 blue.
>
> Purification by std. alkaline lysis, precipitation, CsCl eq.
> centrifugation, 1-butanol extraction of EtBr and dialysis against
> massive volumes of TE.
>
> Now i plan to redo the large-scale preps.
>  Which E.coli strain should I use? Which one is the best to prepare DNA
> for transfection in? Is there a consistent difference or does it vary
> from prep. to prep (growth conditions, media, luck)?
>
> Hope someone can help
>
> -Max







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