Slow cell culture growth? - (Nov/13/2009 )

Hi everybody!

I'm having some trouble getting my cell cultures to grow for electrocompetent cell preps. Here's a rundown:

I transformed my plasmids (Chloramphenicol resistant) into my bacterial strain (US0 deltahisBdeltapyrF- Tetracycline resistant).
From the transformation plate (2xYT+Chlor+Tet) I picked colonies into 5mL 2xYT+Chlor+Tet and let them go over night.
The next morning I dilute that 5 mL culture with 300 mL 2xYT+Chlor+Tet.
It's been 6 hours now and one of my cultures hasn't grown at all, one has about doubled, and one has about tripled.

This is my third attempt. I've tried remaking fresh media and making a new stock of Chloramphenicol.

I've grown up the bacterial strain (minus the extra plasmid) several times for cell preps and they grow up like clockwork in about 2.5 hours to an OD600 of around 0.5. So I'm thinking it's not the strain.

I've had these plasmids in another strain (XL-1 Blue) and they have always grown well. So, I'm thinking it's not the plasmids.

The colonies on the transformation plates look fine and the over night cultures look normal.

The only thing I can think of to try next is switching to LB media. When I make cell preps of this strain without these plasmids I have always grown them in LB. I'm using 2xYT because that's what this protocol I'm following says to do. The two types of media really aren't that different. What's going on here?!?!?!

Thanks!

New thought .... Perhaps they are too far overgrown in the morning. Would starting with a 50 mL o/n culture help?

Hi
Is your plasmids carry the insert and its expression is under control of lac promoter, You may add 1% glucose for O.N growth.
This should give extra-repression of the Lac promoter in case your insert is toxic.
Hope it is helpful
Best
Michael

Thanks for the reply Michael. My insert is under the lac promoter. I eventually need it to be expressed. If I add glucose to prevent the expression of my potentially toxic insert so as to make the prep, am I not merely postponing the inevitable failure of my assay when I need to express my gene?