Trouble going from plates to liquid culture - (Dec/12/2006 )

Hi,

I am trying to amplify a plasmid construct in DH5alpha E.coli cells. The plasmid is Kan resistant and I am plating the transformed cells on LB + 40 ug/mL Kan plates. I get plenty of colonies for my plasmid, and none on the negative control (competent cells alone).

When I try to grow a liquid culture (also 40 ug/mL Kan), the cells do not grow! I tried lowering the Kan concentration in the liquid culture to 30 ug/mL, but still no luck.

Any ideas why this might be happening? What can I do to overcome this problem?

Thanks,
Roshni

can u tell us how do u culture in liquid?
i mean its better to use a large flask even if u are using only 100 ml to get enough aeration to ur bacteria.

I used the same methods I have successfully used before. I grow 10 ml (for miniprep) in a 50 ml tube.