Hello everyone,
I have a frozen stock of DH5-Alpha containing my plasmid which has ampicillin resistance. I want to isolate plasmid by midiprep from this frozen culture but first I want to confirm that they has insert. So what I did is, I streak the frozen culture into ampicillin containing agar plates, after 14h incubation I pick single colonies and transfer them into 5mL ampicillin containing LB liquid media. In both agar and liquid LBs in negative control there is no growth so I thought my ampicillin is working well. However when I do miniprep isolation to confirm it by double digestion before continuing the large culture, I get no plasmid. I tried with different bacteria amounts many times and I still do not have any plasmid. Is there anything wrong with ampicillin? Should I retransform the plasmid and continue but if the problem is ampicillin I think everything will be the same and why I do not have growth in negative controls if the problem is antibiotic selection? I use 100 ug/ml of fresh ampicillin.
Thanks in advance for your answers!
It is not 100uM ampicillin. It should be 50-100ug/ml in final concentration.
by the way, you can contact Noahgen if you want to outsourcing a customized cloning job.
Yes I used 100ug/ml as final concentration, I edited the question thanks for helping.
Hi Dilek.
I also have similar problem with my transformed culture, I would like to read details if you solved this situation. My Competent Cell strain is TOP10 and my plasmids are pUC19 and pcDNA 3.1(+).
Kind regards.
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