I recently bought pVIB plasmid modified e.coli (BL21 strain) and now I want to grow them on LB agar containing ampicillin for selective pressure. I wondered, whether there is a chance due to spontanous mutations that a non plasmid containing e.coli might survive on the amp agar?? Could it ruin my culture?
What can I do to prevent it?
Basically what Artur said:
Be sure to have the original bacterial stock just in case something goes wrong. From this original stock generate one or more secondary stocks which you will actively use.
Additionally, keep an isolated plasmid stock as secondary fall-back option, so you can potentially transform a fresh E. coli stock (or even another species/strain for other uses).
Also remember mutation rates leading to spontaneous resistances are extremely low, so it is unlikely you will encounter this problem. As mentioned by Artur a loss of plasmid or loss of function are far more likely.
It is also recommendable to renew your agar plate cultures regularly from your frozen "secondary" stock. This is not only to decrease the chances of mutations, but also since the bacteria will adapt to growth on agar after some time, increasing lag time and potentially decreasing growth rate in liquid cultures.
Finally, when producing a new "secondary" stock it is always a good idea to isolate the plasmid from the same culture you used to generate the stock in order to make sure the plasmid is still present.
Basically what Artur said:
Be sure to have the original bacterial stock just in case something goes wrong. From this original stock generate one or more secondary stocks which you will actively use.
Additionally, keep an isolated plasmid stock as secondary fall-back option, so you can potentially transform a fresh E. coli stock (or even another species/strain for other uses).
Also remember mutation rates leading to spontaneous resistances are extremely low, so it is unlikely you will encounter this problem. As mentioned by Artur a loss of plasmid or loss of function are far more likely.
It is also recommendable to renew your agar plate cultures regularly from your frozen "secondary" stock. This is not only to decrease the chances of mutations, but also since the bacteria will adapt to growth on agar after some time, increasing lag time and potentially decreasing growth rate in liquid cultures.
Finally, when producing a new "secondary" stock it is always a good idea to isolate the plasmid from the same culture you used to generate the stock in order to make sure the plasmid is still present.
Hi,
for the BL21 strain, being recombination proficient, it is very well possible that a part of the plasmid (possibly only the resistance gene) is integrated into the genome, yielding an selection-resistant strain possibly without the other features present on the original plasmid. This is why for pure DNA work, usually recombination-deficient strains (e.g. DH5 alpha) are generally preferred.
Apart from that, I agree with Mathis, when in doubt fall back to clean stocks (non-transformated competent BL21 cells) and do a fresh transformation with your prepared plasmid.
If you want to be absolutely sure on the presence of your genes on a plasmid (and not in the genome), perform clean plasmid preps (no colony PCR, as it will be positive for genomic integration, too!) on the obtained clones and screen those by PCR, however for routine transformation this would be quite an overkill in my opinion.
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