maybe you see contamination. if colony of bacteria is white and thick, it's contamination.  some contamination grows in agar plate with amp, but i can't grow in broth.

i think Cracking is good way for understanding presence of your plasmid in observed colony.

You should use the same ampicillin concentration both for LB Agar plate and for LB medium. The clonies that grow on LB with 50 ug/ml of ampicillin won't grow on higher antibiotic concentration in liquid media. TOP10 is a good bacterial strain for standard cloning and transformation. Otherwise you can use XL1Blue or DH5alfa strain. I don't know the stratec kit you use,  it's important to use the kit specific for plasmid purification. Usually I use the kits from Promega or Qiagen. Before plasmid purification you can screen your positive clones by PCR.

Is there a chance that your inserts might be toxic for whatever reason? When dealing with toxic proteins, it is my experience that you can sometimes grow certain clones on agar plates, but not in broth.

Another thing, why don't you use Amp @ 50 ug/mL in the liquid cultures?

maybe you see contamination. if colony of bacteria is white and thick, it's contamination.  some contamination grows in agar plate with amp, but i can't grow in broth.

i think Cracking is good way for understanding presence of your plasmid in observed colony.

I had the same problem a couple of times (though I use the same concentration of amp in the plate and in the liquid culture). For some reason I don't know, for same genes I got colonies in the plate but they didn't grow in the liquid. What worked for me was growing the colony from the plate in a small volume of liquid (5 mL) overnight and then transfer to a bigger volume (50 ml).

Thank you all for your suggestions, they are all helpful.

I will match the amp concentrations of the fluid LB.

@Anna: We are screening the colonies, but I think there can be false-positive results very easily

@Alejandro: The proteins are viral but expected to be not toxic. 

@Fereshteh: The colonies are more thin and small (and less) so I think they are not cont, what do you mean by cracking?

@Mariana: We are growing the bacteria in 3 mL o/n cultures. Even if you don't see anything you transfer to a 50 mL medium?

thankfully,

Claus

You have to be careful when using only 50ug/ml ampicillin in your plates when selecting for transformants.  Different E. coli K-12 strains have variability in how naturally resistant they are to ampicillin due primarily to differences in the expression level of their ampC gene.  For some strains you can get away with only 50ug/ml (e.g. DH5 alpha), but others will give you a very high background of non-plasmid bearing colonies at this antibiotic level.  This is why I always recommend that people use 100ug/ml ampicillin in their plates when transforming K-12 strains (even DH5alpha).  So my guess is that the problem isn't necessarily that you plasmid isn't functioning properly, but rather you are getting a high background of untransformed colonies.

I only transfer when I see growth. If the transformation efficiency is low you may need to check many colonies before you find a positive. Is it a three-way ligation what you are trying? I mean two inserts plus the plasmid. That usually gives very low efficiency.

Dear Claus,

I beleive that you are just working with some transformants due to chromosomal mutations. Redo your experiment with well known competent cells and use 100 microgram per ml of Amp.

Regards,

GCKL

I am having a similar issue using ER2738 cells and the pCANTAB5E vector with our insert.  The colonies will grow on Amp plates only when plate in high amounts, which nearly makes a lawn of bacteria.  When I plate 1/10 of the amount, nothing grows.. which doesn't make much sense.  Any time I transfer a colony into liquid culture with the same Amp concentration as the plates, nothing grows.  It is extremely frustrating!  I have also tried using 1:2000 and 1:5000 dilutions of Amp (versus 1:1000) to see if the cells are very sensitive to Amp in liquid culture, but still nothing grows.  Does anyone have any suggestions?

Hi Paige,

My guess is that when you plate a large number of cells, the local concentration of ampicillin is reduced enough to allow growth of a lawn.  This is possible because beta-lactam antibiotics irreversibly bind to penicillin binding proteins (PBPs).  So you are effectively titrating out the ampicillin at the surface of your plate with excess PBPs.  This doesn't work in liquid culture because the rate of diffusion of ampicillin in broth is vastly higher than in an agar plate.  So the resistance you are seeing most likely isn't due to beta-lactamase activity.  

John,

Thank you for the response!  That makes so much sense.  Is there anything I can do so that I can select for Amp-resistant cells (the ones that have the plasmid)?  Maybe other strains of bacteria that I should use that do not have the PBPs?  We are trying to sequence the insert in the plasmid, but we cannot do that unless we can select a single colony and propagate it in liquid culture to miniprep.

Also, if the E. coli cells containing PBPs are the issue, wouldn't I see growth on the negative plates (E. coli cells that were NOT infected with phage containing plasmid with Amp resistance)?

@Claus Georg Gustav Schöning

Why dont you take your transformation mix and inoculate that into your LB ampicillin added media instead of plating it on LB ampicillin agar plate. its worth a try and then isolate the plasmid & check the plasmid with Restriction Digestion or Sequencing.

@Claus Georg Gustav Schöning

another suggestion is try inoculating the colony in LB ampicillin media and grow it at 30°C instead of regular 37°C and let it grow for 24hrs instead of routine 16hours of growth. hope this helps.

Can TOP10 cell take longer than 24  hours to grow??