Lately Ive made a transformation of my cloned plasmid pBAT4 into competent E.coli BL21De3 Plyss. The bacteria cultured on LB agar \ Ampicillin 100ug/ml, 37C over night. The culture showed a contamination with a bacteria or yeast(may be candida) the attached picture shows the contamination .can any one give an advice about the kind of contaminating bacteria and what to do next to have pure e.coli?
And if i can use the same cloned Bl21after purifying it? Or is it better to repeat the transformation?
Hi there,
Pick up a single big clone from your transformation plate and streak it on the same selective media and see if it gives an homogenous population. If yes it's OK if not you will have to repeat the process.
Hi there,
Pick up a single big clone from your transformation plate and streak it on the same selective media and see if it gives an homogenous population. If yes it's OK if not you will have to repeat the process.
The little colonies may be so-called satellite colonies. They are due to the local degradation of ampicillin by beta-lactamase produced by the bona fide transformants.This is particularly the case if plates are left to incubate for a long time. To avoid the problem, you can use carbenicilliin instead of ampicillin. But you can also follow Dominique's suggestion.
Thank you @dominique , i will, and i will also streak both small and big colonies on EMB and on macConkey agar to confirm that it is E.coli.
Thank you @pierre, i have read before about satellite colonies, and i was suspected about it, but you confirmed that, i will use carbencilline...
@dominique and @pierre
I have made vitek for both colonies, they are both E.coli , so the small colonies may be sattelite cells. Thanks a lot.
Best regards
Rana
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