E.coli not growing in liquid LB, but grows well on Agar plates - (Nov/21/2005 )
I am having a small but strange sort of a problem with my E.coli clones. They have the Flag tagged vector in them, expressing my protein, with ampicillin/neomycin selction marker. These bacteria grow well on agar plates supplemented with ampicillin, but when i try to inoculate 1 colony from these plates into LB media (liquid) supplemented with ampiciliin, they fail to grow ?
I am sure I am not picking up the agar only instead of bacteria when trying to inoculte new LB medium. I have tried this twice and my bactreia have not grown in LB .
Some suggestions would be greatly honoured and appreciated.
Can you confirm again is it Amp or other selection-antibiotic what you added. Take fresh and check.
[quote name='Jak' date='Nov 21 2005, 08:17 PM' post='32052']
I know, its is Ampicillin 100 mg/ml stock solution of which i use 1:1000 dilution in my LB. that is what is usually recommended.
Thanks for ur reply
well selection can also be done by diluting your stock solution 1:2000
Try and see if your coli would grow...
If your protein is toxic to the cells this often happens. Ampicillin doesn't last long in liquid media (10 minutes is our lab folklore). Use carbaciillin instead as it is more resistant to the beta-lactamase and will last in liquid culture.
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I'm currently having the same problem.
Transformed E. coli is growing well on Amp plates, but when transferred to 4ml LB + Amp 1:1000 does not want to grow.
I know this might sound like a silly thought, but I would guess the media was still too hot when you added the AMP (amp is pretty sensitive to temperature) so that you are getting poor selection with plated media, and the colonies you see are not really resistant...hence they croak when you add them to broth with AMP
just a thought...also, to avoid propagation of satellite colonies that have kicked out your plasmid, you need to subculture from fresh growth on LB-AMP and not let the plates sit too long in the incubator, or even at room temp
I have had the same experience with some plasmids where the insert is toxic to e coli for some reason. In my experiences, you can grow small volumes well (ie minipreps) however when you try a maxiprep, the bacteria are curing themselves of the plasmid. Tus very small amounts grow and I recovered very small amount of the plasmid. To get around this, I grew up several minipreps and combines the DNA until I had enough to work with. Perhaps try minipreps first and see if you can that to work. If you think you may have a problem with you system (eg amp selection), you can use a control plasmid with amp. I'm sure you can find one in your lab.
Did any of you read my post above?
Check if Amp concentration is same in LB also. After how many hours you got colonies on agar plate? Try using 2X LB. Check if you get colonies if you streak!