Could be toxicity of your insert or bacteriophage contamination causing cell lysis. To discriminate between these possibilities, try to retransform empty cloning vector and see if you can get colonies to grow in liquid culture. If so, your reagents are fine, and the problem is construct-specific, and likely due to a toxic insert. If these colonies don't grow either, then you have a problem with bad LB or phage contamination.

As per information provided by you, it seems on Ampicillin plate you are getting huge number of colonies. I believe then on plate you must be getting satellite colonies too that won't grow in liquid LB + amp. First thing you should do is use 50-75ng of properly digested gel eluted vector for assembly and appropriately use the  gel eluted insert fragments in 2-3X molar ratio excess. Keep pH of LB in plates to 7.3 so that with bacterial growth pH of media should not decrease much to make antibiotic selection ineffective around transformed cells.

Thank you, Kamal. I'll check the pH on my plates.

I also contacted NEB support and received the message below:

 (...) Thank you for contacting us with this issue.  The faint fibrous filament at the bottom of the cell culture tube is usually indicative of the cell lysing, so either there is something wrong with the cells or the inserts are toxic and causing these issues. (...)

I'm then going to try different strains (DH5a, Stbl3 and OmniMAX™2) to see if it was a problem of toxicity with the TOP10.

You may try to transfer a single colony to 1 ml liquid culture media without antibiotics or media with 1/10 antibiotic concentration for 1-2 hour. Then transfer them to the fresh media with appropriate antibiotic concentration. Good luck

Firstly, are you freezing the plates/ storing them at 4 degrees C or below for a long time before picking for liquid culture? Because after a while (about 1 month), the colonies will not be able to grow in the liquid media.

Second, do you use sterile toothpicks to pick your colonies? If so, try to ensure that you do not pick too much agar with the colony, I noticed that hampered my own cell growth. 

As Kamal said, you may have an issue with Satellite colonies. In order to prevent that, I tried to grow the plates in 37 degrees for 2 hours and then at room temperature overnight. It worked for me, I did not see any satellite colonies on my agar+Amp plates, and my liquid cultures were also clean. Then you can pick colonies from this plate to grow in liquid media. Hope this helps!  

Could be toxicity of your insert or bacteriophage contamination causing cell lysis. To discriminate between these possibilities, try to retransform empty cloning vector and see if you can get colonies to grow in liquid culture. If so, your reagents are fine, and the problem is construct-specific, and likely due to a toxic insert. If these colonies don't grow either, then you have a problem with bad LB or phage contamination.

Oxygen..? was liq culture shaking or stationary ?

Best

Was the antibiotic added to the agar in the plates?. The temperature must be not too high to prevent the inactivation of the antibiotic and to make sure the colony is resistant to it. As other researchers, I think you may also have picked a satellite colony that,in fact, will not be resistant to the antibiotic

Bacteriophage issues would also affect other cultures grown in the same shaker or handled in the same lab. Do your colleagues occasionally observe a similar lack of growth?

Do the colonies appear larger than normal after 16 hrs of incubation? if so it may be a fungal contamination. i also encountered similar problems. you should concentrate on the antibiotic, whether the concentration used is Ok, the antibiotic solution should not be too old. u have to re-transform freshly made plates, and use freshly prepared antibiotic solution.  

Besides above possibility like toxic  protein, phage contamination, satellite colonies etc, there is also a possibility of improper washing of your culture tube in which you are growing your transformed bacteria. Sometimes detergents left even after washing. That contaminating detergent may cause lysis of bacteria. Please ensure proper washing of tube also.

if it is not satellite colonies you are picking it could be that your bacteria behave differently towards certain concentrations of antibiotic depending on the form of media, liquid or solid i.e cells are more sensitive to antibiotic concentrations in liquid culture. Try reducing amp to 50microg/ml. It could also as suggested that your insert is toxic and your cells are growing very slowly or lysing o/n 

Hi, Danuta

lentiviral vectors can undergo recombination due to their repeats. Bacteria express different genes in liquid and on the plates. This can trigger the recombination. For some vectors growing culture between 28-32°C already helps. Harvest your cells before reaching the stationary phase.For others, vectors recombination deficient coli (SURE, or STBL strains) can solve this problem.

Last but not least I would like to add that sometimes the antibiotic in the plate (especially Amp), has degraded while in the liquid culture it is fresh, therefore also non-transformed colonies might appear on the plate those coli do not grow in the liquid-culture.  

All the best 

David 

Thanks everybody for such helpful discussion. We have ruled out some possibilities:

-  Ampicillin efficiency/medium contamination:: other strains grew in the same conditions both in the agar and liquid culture;

-  Insert toxicity: inserts are known to be non-toxic;

** This resolved the problem:

I transformed the same reaction product into different E. coli strains (DH5a, Stble3 and OminiMax). Only DH5a and Stble3 had very few colonies in the agar plate, but only DH5a liquid culture grew normally. I was able to recover my construct with the correct insert!

I'm inclined to conclude that recombination was a issue, and cells could not survive in the liquid culture. The reason why I got so many colonies with TOP10 cells is still unclear to me, so I'm not using them for the time being.