Problem with bacterial growth - (Sep/26/2007 )

I have some strange problems with growth of my bacteria and I need some advice.

I have successfully transformed a pcDNA3.1 vector with my gene of interest into XL10 gold and can do an o.n culture of 1 ml from a glycerol stock for miniprep and the plasmid is OK and so on.

My problems starts when to start a bigger culture (200ml) for maxiprep. I prepare a 10 hour 1 ml pre culture and can see some growth. I then put this in 200 ml of LB and grow as usual for around 16 hours but the bacteria won´t grow.

The LB contains 100ug/ml of ampicillin in all cases.

I have tried this twice but no luck and this is the first time I´ve ever seen something like this.

What is going on?

My guess would be that there wasn't any actual growth in the 10 hour 1 ml pre culture. Cell that come out of the frozen stocks are not very healthy or happy. Their growth is not dependable.

I suggest that you streak out your glycerol stock LB + Ampicilin agar plate. Grow that up. Then pick one colony.

Grow that in 2ml LB + Ampicilin for 10hrs. Then use that 2ml culture to innoculate the 200ml culture.

(Although personally I would innoculate the 200ml culture directly with the single colony from the plate.)

You could reduce the ampicilin concentration to 50ug/ml. While perfectly normal, 100ug/ml feel too much for my taste as I normally use only 25ug/ml.