E.coli not growing in liquid LB, but grows well on Agar plates - (Nov/21/2005 )

I've tried reducing the Amp concentration in the LB to 1:2000 and it still did not work.

The plates are definately okay because others in my my lab have been using them without problems.

I don't think the insert is toxic because it is only 39 base pairs encoding for a HIs tag and a protease cleavage site (at the moment I'm modifying a vector before adding my real insert). So the plasmid is efffectively a control plasmid. Someone else in my lab has used this plasmid and had it work.

I'm not going to try carbecellin. First because we don't have it, and secondly because there's no reason why amp shouldn't work.

The only difference between what I'm doing and others in my lab is the strain of E. coli. I'm using DH5alpha and I believe most of the others are using BL21.

I'm going to try BL21 and see if that makes a difference.

Are you getting growth of your ecoli or just no plasmid from your liquid cultures? Your first post is unclear.

Daniel

I'm getting growth on plates but no growth in liquid.

Ok there are three possibilities:

1. You don't have a plasmid and the colonies you get on the LB amp plates are false positive due to low amp concentration. To check this streak out one of your colonies on a fresh plate then scrape the cell and do a miniprep on the scraped cells. If you don't get plasmid then your cells are false positive.

2. There is something wrong with your media.

3. You are killing the E.coli during the transfer. One thing I have seen people do when using a heated loop is not let the loop cool down enough before grabbing the colony.

Daniel

Hi! It is better that you check the following:

1. airation and shaking of your liquid media
2. plasmid is transfered or not
3. concentration of antibiotic
4. temperature of your media and loop
5. your media is fresh or not
6.good luck

Check out what daniel and microdept suggested. One other thing to check is try growing your cells for longer as sometimes the cells can take a little while to start growing especially if they are from an old plate.

As I suggested, an old fashioned control maybe a good idea.

Thanks for all the suggestions. Some of them don't apply, but many might.

I'll try them out and let you know.

Boy this topic was beaten to death!

You may try as follow:
1.) pick 1 colony from the original plate and restreak to new fresh media
2.) if it does grow on the plate then only pick 1 colony to small volume like 2-3ml liquid with antibiotic. shake for half a day then add up to 10ml with fresh media for overnight shaking at 37.
3.) if it doesnt grow on the resubculture plate then it means the colony is wrong. You have to check you cell and your construct.

Good luck