I am trying to clone my gene of interest in plasmid but the colonies appear only on 25ug/ml Amp agar plates and not higher then that. Also when I am trying to grow the colonies in 25ug/ml Amp LB broth, no bacterial growth is observed. What could be the possible reason? And any solutions?
I think a little more context is necessary to really know what's going on, so I have a few questions for you.
-What type of competent cells are you using?
-What is the copy number of your plasmid (or at least, what is the backbone you are using)?
-Have you tried inoculating these clones in LB+0 μg/ml Amp (as a control) and LB+25 μg/ml Amp to determine if they are growing in broth culture? If they are able to grow without selection, I'd say that plasmid isn't being made or they aren't real clones.
-Are you including a negative control in your transformations? This would be nice to include to see if the antibiotic plates are selecting for AmpR colonies.
I'd also try a colony PCR on clones to see if the transformation is actually working to confirm if the plasmid is being made. Make sure to patch these colonies onto a 25 μg/ml Amp plate to save and keep track of the ones you've screened. You could be going through all this work to figure out how to get them growing in culture when in reality, these aren't real clones. With the colony PCR, you can either design primers that are within the gene of interest and somewhere else on the plasmid (preferably where the ampicillin resistance cassette would be to confirm ampicillin resistance from the plasmid rather than random AmpR colonies).
Make sure you're also inoculating your liquid culture with single colonies and using fresh plates to do so. When you're preparing the ampicillin plates, make sure not to add the antibiotic too early. Ampicillin can have its stability issues (carbenicillin is likely a better, and more stable, alternative). I'm wondering if the plates show colonies because the ampicillin degraded (from either old plates or early addition post-autoclaving). I assume you've thought about that issue, but I want to include it just in case.
What I'm including in here is somewhat generic because it's hard to know what's happening without knowing the cells you're using and the plasmid backbone—also, whether the clones are real clones or just random mutants. Hopefully, I've included something you hadn't considered, but if not, it's surely a fixable issue.
I think your colonies are not plasmid contining if they do not grow in liquid culture. We routinely use 50ug/ml AMP on plates. If it is old, often AMP becomes less effective. Try Carbenicillin plates, which are more stable and effective.
With plasmid-carried Ampicillin resistance during cloning it is a bit tricky, since some bacterial strains can produce so-called satellite colonies, these are colonies that do not have the plasmid, but thanks to a close-by Amp resistant (plasmid containing) strains which degrade the Amp in their surroundings can grow in their shadow. The satellite colonies are usually visible as small colonies close to a bigger (Amp-resistant) ones. Given that you mention you have problems re-growing the colonies in Amp (with or without higher concentration) suggests to me this might be the case.
The suggestion for colony PCR from Garrett might help you sort out whether that's the case.
Ampicillin resistant is notoriously "leaky" on solid media. The beta-lactamase enzyme that provides resistance is actually excreted from the bacterial cells and degrades the ampicillin in the surrounding medium.
My guess is that you do not have any transformed cells.
Try again and good luck!
Thankyou for the response.
I transformed the ligated vector into E.coli competent cells and then plated those cells on 50ug/ml Amp Agar plate. Katie A S Burnette
Gregory Dressler I tried with fresh plates but the colonies are still not growing in broth.
Garrett Reynolds
I am using E.coli DH5a competent cells and pcDNA3 vector backbone. I have used control for transformation, transformation is not the issue. And also regarding the Agar Plates, I tried with freshly prepared plates but still the problem remains same.
I tried Amp concentrations ranging from 10ug/ml to 50ug/ml but the colonies are observed only till 25ug/ml Amp. And those colonies are not growing in broth with 25 or less Amp concentration.
Even I am not getting what actual problem is.
Since 100ug/mL is the standard working concentration of ampicillin, it is possible that these colonies are just non-recombinant DH5a which are susceptible to ampicillin when it is at the working concentration. And because in your case the concentration was lower than this, these susceptible cells were able to grow. As other people have already mentioned, this may have been compounded by ampicillin degradation.
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