Inability to grow plasmid in culture - Molecular cloning (Jun/13/2007 )
Hi there
I've got this plasmid which I used for transforming TOP10 cells (invitrogen). My problem is that the transformed cells seem to grow on an Amp plates but do not grow in culture containing LB+Amp. I haven't got much info about the backbone of the plasmid -I only know it carries the targeting arms of the gene I want to knock out and some unique restrctriction sites. I suspect though that the backbone is from pBSK (II) (-). Any tips I could try for getting my plasmid to grow in culture?
Many thanks
I've had this problem in the past as well. In my experiences, the concentration of ampicillin in plates is never as high as in liquid culture, even if you add the same amount. Therefore, I usually add a bit more ampicillin to my plates to get the desired concentration.
Is it possible that this is why they grew on plates alone, but not in liquid? Are the colonies large on the plates (growing well)? I would try lowering the concentration of ampicillin (or try a gradient to optimize and save yourself some overnights) in the liquid, and streaking those colonies so you don't lose them. Additionally, is it possible that the colonies that grew don't have your plasmid, but were growing due to the lowered ampicillin concentration? In this way, they would not grow in liquid. Are you sure the promoter is recognized by the TOP10 cells (if not, they might pop up on a plate in low concentration, but not in liquid, although I doubt this is your problem).
A few things to think about if nothing else.
I've got this plasmid which I used for transforming TOP10 cells (invitrogen). My problem is that the transformed cells seem to grow on an Amp plates but do not grow in culture containing LB+Amp. I haven't got much info about the backbone of the plasmid -I only know it carries the targeting arms of the gene I want to knock out and some unique restrctriction sites. I suspect though that the backbone is from pBSK (II) (-). Any tips I could try for getting my plasmid to grow in culture?
Many thanks
As cheamps suggested, you could be inoculating satellite colonies which will not grow in solution.
Try to make plates with higher conc. of ampicillin and retry minipreps or try picking 4-5 colonies and hope for one for them to grow.
Try to make plates with higher conc. of ampicillin and retry minipreps or try picking 4-5 colonies and hope for one for them to grow.
Amp plates are best to be made fresh (1week max), because the antibiotic got degraded quite fast. While compare to culture solution, you usually always made fresh, so the colonies that grow on your plate may not contain your construct. When I use Amp plates, I have to use all controls (competent cells only, mock-transformed, uncut DNA, cut DNA, you-name-it). Also, due to the degradation of Amp and the nature of Amp antibiotic effect on bacteria, it is easy to get satelite colonies --> more problems with picking true transformed colonies. I usually use 100-200ug/ml Amp for both plate and solution, and I don't plate for >16h. It may help.