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questions about TOPO cloning - (Mar/03/2009 )

hi, there. I meet some problems when doing TOPO cloning. I ligated my target DNA into pET101 TOPO vector and transformed it into TOP10 E. coli competent cells. They were growing very well on LB plates with 50 ug/ml ampcillin. But when I randomly picked some of the single colonies (I picked 8) and restreaked it on the new plates with the same concentration of ampicillin, they did not grow at all after one night!!!! :unsure: I don't know where I am wrong and I did it twice. The results are similar. It can grow after two nights but the cells did not look quite healthy. When I innoculated some of them into liquid medium with same or even less concentration (25 ug/ml) of ampcillin, they ceased to grow. I was quite shocked by it and want to ask the professionals here if anyone had the same situation before and how I can solve this problem. Thanks very much for your reply in advance!!!

-zx0819-

zx0819 on Mar 4 2009, 04:41 PM said:

hi, there. I meet some problems when doing TOPO cloning. I ligated my target DNA into pET101 TOPO vector and transformed it into TOP10 E. coli competent cells. They were growing very well on LB plates with 50 ug/ml ampcillin. But when I randomly picked some of the single colonies (I picked 8) and restreaked it on the new plates with the same concentration of ampicillin, they did not grow at all after one night!!!! :unsure: I don't know where I am wrong and I did it twice. The results are similar. It can grow after two nights but the cells did not look quite healthy. When I innoculated some of them into liquid medium with same or even less concentration (25 ug/ml) of ampcillin, they ceased to grow. I was quite shocked by it and want to ask the professionals here if anyone had the same situation before and how I can solve this problem. Thanks very much for your reply in advance!!!

I faced this sometimes (not TOPO cloning) but I realize that I just got this situation with Amp, not the other antibiotics. Maybe the degradation of Amp by resistant cells (correct transformants) is fast, then when Amp concentration is reduced locally, and without the need of resistant gene, the cell discard the plasmid (the "satellite colonies" phenomenon) and they might grow better than the transformed cells and you picked them. Of course, they can't grow in the fresh Amp medium. Normally when I performed my experiment again or picked the cell to the master plate sooner, this didn't happen. Just my opinion.

-Quasimondo-

Does ur vector contain Kan resistance gene as well? perhaps could use tht instead. the TOPO vector i have contain both Amp and Kan resistance gene.

IN any case, for the Amp problem, try going up to 100ug/ml.

-hanming86-

hanming86 on Mar 4 2009, 09:45 PM said:

Does ur vector contain Kan resistance gene as well? perhaps could use tht instead. the TOPO vector i have contain both Amp and Kan resistance gene.

IN any case, for the Amp problem, try going up to 100ug/ml.

He's right ^^

-Quasimondo-

hanming86 on Mar 4 2009, 08:45 PM said:

Does ur vector contain Kan resistance gene as well? perhaps could use tht instead. the TOPO vector i have contain both Amp and Kan resistance gene.

IN any case, for the Amp problem, try going up to 100ug/ml.

No, my vector does not have Kan resistant gene. But I will try to increase the concentration to 100 ug/ml.
Is the Amp resistant gene also applicable to the antibiotic Carbenicillin, which is said to be more stable than ampicillin?

-zx0819-

zx0819 on Mar 4 2009, 07:28 PM said:

hanming86 on Mar 4 2009, 08:45 PM said:

Does ur vector contain Kan resistance gene as well? perhaps could use tht instead. the TOPO vector i have contain both Amp and Kan resistance gene.

IN any case, for the Amp problem, try going up to 100ug/ml.

No, my vector does not have Kan resistant gene. But I will try to increase the concentration to 100 ug/ml.
Is the Amp resistant gene also applicable to the antibiotic Carbenicillin, which is said to be more stable than ampicillin?



I have heard of Carbenicillin . Yes they are more stable but i am not sure about the amount that you need to use.

-hanming86-

zx0819 on Mar 4 2009, 12:41 AM said:

hi, there. I meet some problems when doing TOPO cloning. I ligated my target DNA into pET101 TOPO vector and transformed it into TOP10 E. coli competent cells. They were growing very well on LB plates with 50 ug/ml ampcillin. But when I randomly picked some of the single colonies (I picked 8) and restreaked it on the new plates with the same concentration of ampicillin, they did not grow at all after one night!!!! :) I don't know where I am wrong and I did it twice. The results are similar. It can grow after two nights but the cells did not look quite healthy. When I innoculated some of them into liquid medium with same or even less concentration (25 ug/ml) of ampcillin, they ceased to grow. I was quite shocked by it and want to ask the professionals here if anyone had the same situation before and how I can solve this problem. Thanks very much for your reply in advance!!!


I always have this sort of problem with amp - I generally go up to 200 ug/ml.

-microgirl-

Carbenicllin can be used interchangeable and similarly to Ampicllin with the added benefit of being more pH stable, but a little more expensive.

-NemomeN-