I have transformed around 1000 ng of my circular plasmid into E. coli BL21 (DE3). However, there was no colony but a transparent bubble-like 'colony' formed at the bottom of the plate (As attached) . May I know what is it and how to overcome this?
I think there is nothing wrong with my competent cells as the transformation of positive control (blank vector) was successful.
In picture A those are just air bubbles that formed in the plates. In picture B it is hard to see what you have, the image is not very clear.
Michael J. Benedik Thanks for your reply! If this is air bubbles, may I know what is the possibilities of unsuccessful transformation for isolated plasmid? Because for the positive control, I can get the colonies.
I can't diagnose why you aren't getting real colonies. It might be that you don't actually have plasmid DNA, did you confirm on a gel that your plasmid DNA is good?
Also how many colonies did you get with the positive control transformation? It may be your competent cells are not very good.
I already sent my sample to sequencing and validated the recombinant plasmid through the gel. Attach is the plate for the positive control. I use the same amount of DNA for both control and my recombinant plasmid.
Your competent cells are really poor. With the amping of DNA you are using you should have the plate covered with colonies, at least 10,000 or more
I see, thank you for the input! Should the recombinant plasmid also has at least several colonies since my control can give the result even there is not much since my insert is around 500 bp only?
Maybe, but you got so few colonies with the positive control that if the quality of your recombinant plasmid is not as good, you might just not be getting anything.
Have you checked the quality of the plasmid? You should only need to use a few ng (not 1000 ng) for a transformation.
I checked the quality of the plasmid on a gel and nanodrop. The plasmid seems fine on a gel and based on the nanodrop reading. For your information, I did not do the RNase treatment during plasmid isolation. Does it affect the transformation step?
Skipping Rnase should not impact the transformation. Using 1000 ng is a ton of DNA, you may want to try using a smaller amount. The colonies you are getting look really nice so if the goal is just to have your plasmid in culture then it looks like you succeeded. I agree that the efficiency is quite low. Are your competent cells starting to get a bit old? They generally stay good for about a year at -80.
Amy Klocko, thank you for your reply. I will try to reduce the concentration of DNA. And yes like you said, my competent cells are getting old. Perhaps, I will need to prepare the new one.
Hi, it has been a long time since the issue has been resolved, but I still think it's worth sharing.
I did not experience the same issue again when doing transformation using the newly prepared competent cells.
Thank you for all the replies.
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