I am doing transformation with several plasmids into E.coli cells. I have used pUC19 plasmid, GFP plasmid and our expression vector plasmid in different transformations and I have used two different antibiotics, Ampicillin and Kanamycin. I expect colony formation for all transformed cells but cells do not form colonies. They are growing but in a different way, like in the picture below and when I take cells from petri, inoculate into LB Broth with suitable antibiotic and do midiprep extraction next day, I got no plasmids. I am dealing with this problem for one month and I have no other solution anymore. Has anybody faced with this kind of problem?
Hello,
Because of the result obtained on the 2 culture media, I suspect the antibiotics. Maybe your antibiotics aren't working.
Have you tried culturing your host cells before transformation in the presence of the antibiotics to confirm that your antibiotics are working?
Hi Sofiane,
I did not try that, Kanamycin might be the problem because it has been opened since 04.2018, it is too much time for antibiotics to be open but Ampicillin powder is newly opened and stock solution also is prepared fresh from powder, these pictures belong to Amp plates. Do you think that too much cell concentration can cause this?
Hi Elçin,
Since the antibiotic selects only transformed cells, I don't think that transformation can be so effective in giving all this culture, moreover you said that inoculation on LB did not give anything, so it is not transformed cells.
What volume did you put on the Petri dish? If you suspect a significant bacterial cell load try seeding with 0.1 mL of suspension.
Your plates look similar to LB without antibiotics as you are getting a smeary sort of lawn growing.
Your Kan is likely expired, and Amp can breakdown quickly. So, you're likely losing selective pressure. That would explain why you aren't getting any plasmid back out after your liquid culture.
Also, if the antibiotics are added when the medium is still hot the antibiotics will break down.
I would suggest making up fresh solutions of your antibiotics, filter sterilizing them, making small aliquots, and storing at -20. Add the antibiotics to liquid cultures right before culture inoculation (don't store liquid LB with antibiotics, it breaks down).
Hope this helps!
I think it’s too much concentration, You should try to dilute the cells.