My E coli culture has started showing some clumps after repeated innoculations and I suspect it is contaminated. I do not have another culture containing my plasmid.
First do as Jose suggests make sure that yu selection is working. The only probelm will be if there are other plasmids in the contaminating strain but just extract the plasmid and do a restriction digest and check if you get more bands then you expect (use a restriction enzyme that cuts at least twice). If you have extracted more than one plasmid, you can do a transformation of the extracted plasmids and grow new O/N cultures and test witch colony has your original plasmid, but at this point it might be quicker to transform with your original plasmid DNA.
Jose, my palsmid has ampicillin resistance gene. But each time i re-inoculate my plasmid containing culture in fresh medium, i add ampicillin to prevent contamination. Since the culture got contaminated inspite of this, perhaps the contaminating strain itself has ampicillin resistance.
Gustaf, thanks for the suggestion. I will try restriction digest and see what i get.
I would do exactly what Gustaf suggests. I would also make up a new amp stock (filter sterilized 50 mg/mL stock; working concentration of 50 µg/mL in your culture). Sometimes it's the simple things, if we're lucky.
Maybe also a more "primitive" method like subculturing in a selective medium O/N could do the work. Differential media for Enterobacteriaceae like MacConkey agar, Violet Red Bile Agar or the more sophisticated Chromocult E. coli agar are suitable. Then you can check for AMP resistance and the presence of the plasmid.
I agree with Vagelis, that suggest you a simple subculture in a differential medium (MacConkey, EMB are the easiest ones to prepare but as Vagelis said, there is also the Chromocult that is very specific to detect E.coli) ON and then you can select for ampicillin resistance, since your contaminant is also resistant to the antibiotic. Otherwise, you risk isolating the plasmid (if there is any) from the contaminant and although the band number and size is a very good indication, it doesn't help to get rid of the contaminant. I would also prepare ALL the media and the antibiotic stock again, it's like Christina said, sometimes the simplest things ruin a week (or more) of work.
Best of luck
In fact to isolate a plasmid the culture has to be very, very (if not ultra) pure. Secondly you must be sure as to what you are looking for to be present on the plasmid, which should be absent once the culture is cured by any means. Finally then try to isolate the plasmid. Well it is some thing like trying to prove Koch's postulate with the plasmid isolated (which is the next step). You will not only be able to isolate the plasmid but get the desired results.
You could just streak it for single colonies, that would be the simplest and you would also see if you have contaminants or not.
But you can also do a mini prep and retransform. Unless the contaminant also has an Amp resistant plasmid that will work fine. I world reconfirm the plasmid is correct by restriction digest or PCR
Grow the cells on a plate and since you know the plasmid, you can select single colonies having the plasmid by Colony direct PCR
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