competent cells advice?

[D.K.]

ms2241 at columbia.edu (Melanie Sohocki) wrote:

>         I am having some insert instability problems with my subcloning. 
>In the past, I have used the SURE cells from Stratagene to combat this 
>problem, but both strains of these cells are resistant to kanamycin, the 
>same marker on my plasmid. Unfortunately, I cannot simply change vector, 
>because it is for a yeast-two hybrid application downstream. Any advice 
>would be appreciated!
>

Just an idea: Avoid liquid cultures (o/n cultures in particular) by all 
means. All problems with insert instability/toxicity are greatly reduced 
when one tries to do as much as possible on plates and as little as possible 
in liquid. 

Don't ask me why because I don't quit understand it, but I know it
works. 

DK