Hey,

im not too sure why that is happening with the DH5alpha, are they old?

if you still have the plasmid from the positive clone with no mutations I would try to clone it into different E. coli strains, such as Neb10beta. Transformation should be very efficient so only use 0.1ng to ensure 1 plasmid copy per cell. You could even try multiple strains and test whether the problem arises from the plasmid sequences (maybe it has an ORF somewhere that the bacteria can read and somehow increase mutation possibilities?) or it could just be strain-dependent and be sorted using a different one. good luck!

If you are getting these deletions in your reading frame, that is suggestive that the product of the ORF is somewhat toxic on E. coli and you are selecting for mutants.

I would try a different host just to see if that helps, but more importantly I would not grow for nearly so long. Try to pick your colonies when they are smaller and be sure not to grow the culture too long (not overnight just long enough to get good density cultures.

Or you could try growing at 30C or lower, but again maybe for 24 hours, not for 2 days. The longer time you incubate the greater the likelihood that a mutant will take over the population.

Try putting the insert under control of an inducible promotor. That can reduce the possibility of accidentally selecting for loss of expression of a toxic protein.

Michael J. Benedik and Bruno Salomone Gonzalez de Castejon A new host would be an easy thing to try, good thoughts. The DH5a cells are ~4 y.o. though we have made ~30 constructs this year with no issues except this plasmid.

I'll try less grow time, which I hadn't considered as I was focused on making sure there was sufficient replication time for the large, low copy plasmid.

I appreciate your thoughts and ideas, thank you!

Katie A S Burnette That's a good thought, though I think it would be difficult in this situation as this plasmid contains 4 separate genes in the ORF (with p2a fragments to cleave them apart). Without knowing what genes/aspects are toxic, it could be quite a project to redesign this way.

Going to backpocket this thought for future issues, thanks!

some mammalian sequences is hardly replicated in E. coli. Also, large plasmids makes replication difficult. Try to use E. coli strain appropriated for replicating mammalias sequences such as XL10 gold.

For anyone who has stumbled across this post- I just powered through and had success.

I retransformed the confirmed correct DNA and started maxicultures of 4 different bacterial clones. I grow for our standard 2 days at 30'. I just took a small amount of culture and miniprepped it before pelleting the maxi culture and freezing it. I whole plasmid sequenced the miniprepped DNA and happily 2 of the 4 came back without any base indels so I proceeded with maxiprepping the corresponding pellets.