1) Make sure the antibiotic stock is fresh and/or stored at -20oC, and after the frozen stock has thawed vortex it to get the antibiotic into solution (ampicillin will settle to the bottom of the tube as a "clear" layer under the water).

2) I would first streak the glycerol stock out onto a fresh Amp. plate, and then pick a colony from that plate to start an O/N culture. The next morning I would then centrifuge the O/N culture, remove the supernatant/media, and resuspend the E. coli pellet in media with fresh Amp. The O/N culture "media" will contain lots of beta-lactamase which will rapidly degrade any fresh Amp. removing the antibiotic selection pressure.

One problem could be that your culture grows too thick when starting from the glycerol stock. This could result in incomplete lysis and poor plasmid yield. What media do you use? LB, SB or something else? I have had a similar problem and it was solved by either just using a smaller volume for a miniprep (1ml) or by freezing the pellet after ON incubation (this will facilitate lysis). Make sure you get a transparent solution when adding the lysis buffer. If it is cloudy, it is a sign of incomplete lysis of bacteria.

Good luck!

Did you maintain the drug pressure of the selection marker of your plasmid? This will avoid the growth of any bacteria without it. If your plasmid express a toxic protein (even if it is inducible) and you did not use selection drugs during the growth, plasmidless bacterias can be selected for their faster growth rate. Additionaly, did you check that your final culture is E. coli, could you have some contamination problems? (This would be even more weird, it is difficult to imagine something able to overgrowth E. coli).

1) Make sure the antibiotic stock is fresh and/or stored at -20oC, and after the frozen stock has thawed vortex it to get the antibiotic into solution (ampicillin will settle to the bottom of the tube as a "clear" layer under the water).

2) I would first streak the glycerol stock out onto a fresh Amp. plate, and then pick a colony from that plate to start an O/N culture. The next morning I would then centrifuge the O/N culture, remove the supernatant/media, and resuspend the E. coli pellet in media with fresh Amp. The O/N culture "media" will contain lots of beta-lactamase which will rapidly degrade any fresh Amp. removing the antibiotic selection pressure.

I had the same problem. Everything will be fine if you streak in a plate before, and then pick up one colony.

I agree with Gary, but I would add 1-2 more steps to guarantee less time wasting:

1- Streak your frozen bacteria O/N, the reason you have plasmidless bacteria on your O/N plate is probably because of contamination. Other antibiotic-resistant bacteria grow with your E. coli. and that is a common result for contamination.

2- Pick up few colonies for colony PCR, use a tooth-pick to touch the colony on the plate then rub it in the PCR tube, and then through this tooth-pick in the tube with the medium and the antibody for the O/N culture. Do this for several colonies, I usually do this for 2-4 colonies.

I dont have to wait overnight to see which colony is correct or not, the colony-PCR would answer me in few hours, then on the next day I choose those which already gave me a positive result from the RT-PCR for plasmid extraction.

Additional step:

3- After you extract your plasmid, do treat it with restriction enzymes (according to your plasmid map) and check the size of the bands cleaved. this ensures you have the correct plasmid extracted from the correct bacteria.

4- Good luck.. I am now going to do the same steps for my E. coli :)

Hello there.

1) Check that your antibiotic on the actual agarose plate and culture is active. Repetitive freeze/thawing may pose a de-activating factor for your antibiotic hence none of your plasmid containing colonies may be favoured.

2)The above may have as a result that you have your culture contaminated by otrher faster growing bacteria that outgrow your desired ones.

3) As many people mentioned, make some fresh antibiotic, make new media, reculture and see what you get. I would certainly restreak some of your frozen glycerol stock on a fresh plate and pick colonies (about 10 will be fine). Do a colony PCR (FASTER) or get minipreps out of the 10 picked colonies and perform a suitable digestion according to your specific digestion map for that desired particular plasmid. Se which clolonies contain theplasmid and continue with them only.

Best of luck,

Nikos

From fundamental microbiology point of view, plasmid loss is one of natural characteristic of plasmid that is not being generated to the next offspring cells. So, it's not uncommon phenomenon. ArgR plays important role in site-specific recombination maintaining plasmid content in a cell.

Therefore, some factors mentioned previously such as single colony, fresh antibiotic required when one makes a glycerol stock.

I am not sure if anyone has mentioned it is using an 8% glycerol stock rather than higher glycerol. We have found that at higher glycerol concentrations that the surface thaws and re-freezes. This tends to select for cells without plasmid. If you are working with a plasmid that is for expression and in the expression host you may want to test how well you are maintaining the plasmid to begin with. If it is IPTG inducible LB contains significant amount of an inducer (lactose or related sugar) and anytime you grow it in LB you get some leaky expression. In that case use a minimal or fully defined media containing glucose (shuts off lac), or add glucose to LB. Finally, if your selectable marker is amplicillin try to switch antibiotics Kan works better. Ampicillin is hydrolyzed rapidly by bacteria carrying amp resistance and, in fact, there is no selection above about 0.1OD 600nm. Carbanicillin is better but not by much. You should remember this also with growth on plates. Do not let your bacteria over grow or switch to minimal plates (Bill Studier has a very good repressing media MGD I think its called). Unfortunately this is a bit rambling since its is not clear whether its due to a toxic insert, large or potentially recombination prone insert or something else. This could help localize the type of problem and solution.