Thank you, it was done with selection.

I do not fully understand how the untransformed bacteria survived the selection. I assume it was similar to how satellite colonies form.

The "no DNA" transformation plate was empty.

I initially thought it was wild type gDNA leftover from dead bacteria that was serving as the template for the PCR, but the wild type gDNA persisted after many passages.

Hello Gen

see the map of the plasmid, if there is an antibiotic resistance gene you can do selection!

Thank you, it was done with selection.

I do not fully understand how the untransformed bacteria survived the selection. I assume it was similar to how satellite colonies form.

The "no DNA" transformation plate was empty.

I initially thought it was wild type gDNA leftover from dead bacteria that was serving as the template for the PCR, but the wild type gDNA persisted after many passages.

Are your no template controls both negative in the pcrs.? There may be contamination in a common reagent like the water

Try a different taq polymerase in case there is homology with dna left in the taq after a poor purification by the manufacturer

As Paul Rutland mentions, did you do all the correct controls and did you get the correct profile from the negative control? It could be the issue is with the PCR. Assuming it is not, are you certain the plasmid is NOT replicating in your host, and are you certain there is not alternate integration sites?

Paul Rutland

There is more information: This only seemed to happen when I used a "weak" antibiotic selection with high spontaneous resistance rates.

For a different transformation, I also used a "strong" antibiotic selection (that has almost no spontaneous resistance rate) and this issue did not occur. The "Wild type only" pcr was negative when using strong antibiotic selection, while the "transformed only" pcr was positive.

The PCRs and transformations were all done in parallel. So I don't think there was contamination in my reagents.

Michael J. Benedik

I suppose there could be an alternative integration site. However, then my "transformed only" pcr should be negative. As one of the primers is specific to that integration site. And I did have a negative control for pcr as well that was "as expected."

I think you have answered the question then, if it works with the "strong" antibiotic but not the weak one, then you most likely are getting mixed cultures and have background cells that are not resistant. Even well separated colonies can have a small amount of cross contamination, in fact there are likely to be background cells present in an area of the plate without any colonies, unable to grow but not really dead.

Best solution is to either increase the selection pressure or preferably streak out and get pure cultures.

Michael J. Benedik

Does it seem likely these untransformed bacteria could take over the culture? I worry they could become spontaneously resistant entirely.

I am just saddened that I must wait several more weeks for the colonies to regrow, and even then it's possible those could be mixed too. This bacteria is clumpy.

Thank you for the additional information Gen Gi . I think that Michael J. Benedik is correct. It is both a strength and a weakness that pcr is capable of amplifying even tiny amounts of DNA contamination

Several weeks to grow seems a long time Gen Gi . I wonder if part of the problem is that the antibiotic is losing its activity as time passes allowing the untransformed cells to grow in which case picking colonies earlier might help

Paul Rutland

It's possible the antibiotic degrades, but the bacteria grows slowly -- it is not possible to pick earlier.