I'm not totally sure I follow your experimental design but it sounds like you have a gene that expressed off of your plasmid very reliably previously that doesn't seem to do so anymore after prepping new aliquots of it.

You can have significant recombination occurring in your bacterial propagation strain, especially if the plasmid has repetitive sequence. To combat this, I'd rely more on re-transforming fresh for each new prep (as opposed to re-streaking from a glycerol stock) and to consider changing to a low-recombination bacterial strain like NEB Stable or Invitrogen Stbl3.

I like to fingerprint new plasmid preps with restriction enzyme digestion that produces a known expected banding pattern. It's a good way to ensure that you're not getting significant genomic DNA contamination in your prep or that you haven't selected a clone with major plasmid rearrangements.

Another thing to consider is your 293T cells. Are you transfecting cells from the same batch of stocks that worked well for you previously? Are they in good health and at optimal density at the time of transfection? Cell lines can be much more variable from experiment-to-experiment than is appreciated.

Hello Govinda,

Thank you for your input!

I agree with the possibility of recombination. I just find odd the fact that it behaves so differently since the sequence is identical. I am currently sequencing the promoter to see if there are mutations occuring there.

Yes the batch is the same. You have been experiencing some contamination problems up until recently, but my cells are growing fine and healthhy without antibiotics as well, so far at least.

I always transfect around 60% confluency, using Lipo 2000, changing the media after 5 hours.

Thanks again for your input!

If you think it is your new plasmid batch, then I would remake it. But be sure to streak out from your frozen stock to get single colonies and pick a well isolated colony to grow. In fact you might prep 2 or 3 different single colonies. That way you know each stock is clonal and not contaminated and one of them should behave consistently.

Have you checked the plasmids on agarose electrophoresis against original plasmid? It seems likely that that particular purification may not have gone completely to plan and your DNA quality is not as good. Supercoiled DNA is the best for transfections, if you have less of this present compared to previous preps then this may explain the differences seen.

As suggested I would re-prep the plasmid, transforming from the original (if any left) and re-analyse.

Hey,

as the others pointed out re-prepping is always a good option. However, given that your sanger sequencing results are identical between batches, I would rather regard the cells being the origin of your issues, as Govinda also mentioned above.

You mentioned a recent contamination, of what type? Did you check your cells for mycoplasma or latent fungal contamination? In what passage number are you operating? Did you give your cell line some time to adapt after uptake from cryopreservation? Did you change any media ingredients or formulations? Do you use serum, what about your serum stock?

Just a few thoughts from the cell perspective, I hope that might help in any way.

Best regards

Sebastian