1 reply to this topic

[Luria Bertani]

Hi there,

My protein appears to be toxic and so I get few colonies and they are quite small when I do a plasmid miniprep using BL21 (DE3) cells.
I strongly suspect leaky expression of toxic protein is the cause, so I want to collect plasmids from something that suppresses basal expression levels. Perhaps a strain that has pLys.

Problem is, if I do my transformation and plasmid prep in these strains, these also have pLys plasmid as well, so won't that give me my plasmid plus pLys plasmid contamination???

[pDNA]

i would never do plasmid isolation from expression strains like BL21. BL21 is recA+, therefore tends to multimerization of plasmids. Plasmid isolation from those strains gives low quality plasmid DNA. Additionally, when you use pLys strains you will get a mixture of plasmid DNA.
I would not recommend that! Do plasmid isolation in cloning strains that are historically used like DH5a, JM109, ...

Regards,
p