Cloning toxic gene - (Oct/10/2007 )

Hi,
I am working on a project trying to clone a gene that is toxic to E. coli. Specifically, I'm trying to correct a mutation using site-directed mutagenesis. After the SDM PCR reaction, I've tried transforming One Shot Top 10 & NEB turbo E. coli. I get various rearrangements, additional mutations, and inversions. I tried using Epicentre's ABLE C & K E. coli, but my plasmid is kanamycin resistant and so are the cells....

Any suggestions?

Thanks!

get tighter controlled promoters for your gene. Avoid using lacZ promoter.

grow your cells at a lower temperature 28 celsius (including recovery). Lower temperature, means lower gene activity, means less toxic gene being expressed. For plasmid extraction purposes, don't culture your cells to high density. Keep the OD low (OD600~0.3-0.5), compensate by growing larger volumes. (all culturing work conducted at low temperatures ~28 Celsius)