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

T. Max M. Soegaard tmms at mbio.aau.dk
Tue Nov 7 07:49:13 EST 2000


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






More information about the Methods mailing list