As in the past I have transformed my bacteria with a cDNA plasmid vector, and I seed them into dishes. First thing that I noted that bacteria grew just in the first 2/3 of the dishes, where I poured the 50ul of the transformed solution, and in the second 1/3 where I started to spread but not in the last 1/3 where they should be very few. Anyway, I made a mini and a maxi that were not particularly cloudy even if I could feel the smell of bacteria. I started my extraction from the 100mL of bacterial solution and with great surprise I didn't get any DNA. My quantification with the nanodrop was negative.......!!!
Anyone could give me a right explanation? I want to tell you that for some reason I got the bacteria from another lab, and since I was not able to store the bacteria immediately at -80 I stored them in dried ice at 4C. However, the day after I saw that the dried ice was totally evaporated. Could the bacteria be dead?
If the bacteria grew on the LB plate which I assume contains a selection antibiotic (ampicillin or kanamycin being the popular one), then the bacteria are not dead. If your mini and maxi were not particularly cloudy and your plasmid is low-copy, then that might explain why you don't get any cDNA. It is hard to pinpoint exactly where it went wrong in the transformation protocol if you don't have a positive control you know works.
What kind of competent cells do you use? Are you sure that your selection antibiotics matches the resistance in the vector. Normally competent bacteria should be stored directly at -80°C and slowly defrosted on ice.
I am absolutely sure that the plasmid vector leads the resistence for Amp as I have already and several time used this bacteria when I was in the lab from which I received now the bacteria. The difference is that when I was in Cambridge I got hundreds ug of cDNA plasmid now -somehing. Making a research on teh website I have seen that it's possible that bacteria alreadi died are able to grow in the competent media...and you are not able to see if they are dead or alive. It sounds strange. However in my case I don't know exactly what's happening, as in the amp agar dish I have a growth even if not the special one, just in the firs 2/3. whereas in the last 1/3 not....!!! The bacteria are Top10.
Happened to me many years ago that I got a bacteria stock which had somehow acquired the antibiotics resistance, but were not competent. I changed to another stock of different DH5alpha, made out of them chemically competent myself, problem solved.
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