I have a pHelper plasmid that is 13kb. I have successfully gotten it to grow using DH5alpha cells in the past. There usually weren't many colonies and I would either need a large volume of culture or I would have to grow the culture longer in order to get enough material to do a maxi prep.
Recently, we have been unable to get any colonies when we do the transformation. We tried electroporation instead of heat shock with two different competent cell lines. Both have yielded a lot of tiny colonies at a 1:10 dilution or no colonies at all at a 1:100 dilution. I've tried picking colonies to grow up and send for sequencing. Out of ten colonies I sent for sequencing, only two had the full length plasmid. The others had truncated plasmids or larger plasmids containing an E. coli transposon. I tried growing cultures from the two colonies that had the full length plasmid, but hardly anything grew.
Has anyone run into this sort of issue in the past?
Hi Morgan,
I am not sure what plasmid you use, however what you describe sound that your plasmid might be toxic for the cells so that you get only small colonies (or none colonies et all).Toxic plasmid are also often unstable what might explain your truncated/mutated plasmids ( due to selective pressure).
If this is the problem you might try lower temperatures for longer times for growing the (20-30°C) - for the plates and for the liquid culture.
There also cell lines expecially desiged for propagating toxic and/or unstable plasmids.
I hope this helps
I agree that sounds like either a toxicity issue or something else that's being selected for (like higher antibiotic resistance gene expression or plasmid copy number, which can happen if expression of the antibiotic resistance cassette is very poor due to whatever reason). But the most likely answer is toxicity.
If you found multiple clones with transposon insertions in different places, were there any patterns to where the insertions were clustering? If so, that might give you a clue where the problematic region is. I had an issue where I was cloning a gene that was toxic to E. coli once. None of the colonies grew when subcultured at 37°, and at 30°C it took 2-3 days for colonies to grow, but it turned out all of the ones grown at 30°C had IS1 insertions which disrupted the coding sequence of the gene I inserted. The insertion sites were all different, which told me expression of the gene was being selected against multiple times in different events.
It's hard for me to guess exactly what's going on here though without seeing the intended sequence and the sequences of the clones actually obtained.
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