Hi
Just wandering if anyone else is observing a rearrangement of a plasmid, with partial loss of the plasmid region that doesn't contain Ori, in plasmids that contain two or more of the same/similar sequences (promoters, for example).
I had no issues before but I'm lately observing a loss of plasmid regions when scaling up from mini to maxiprep.
Please let me know if you have a suggestion how to address this.
Best,
Jana
Is the plasmid deletion you are seeing explainable by recombination between the two similar regions on the plasmid? Then as alluded to by Kristin M. Adams there may be an issue with which strain you are using (and whether or not it is recombination proficient or deficient).
@ Kirstin
I'm using Top 10. I'm not too familiar witj this strain.
Before I worked with the same two regions im DH5ALPHA and had no problems. Not sure if it was due to the strain, or pure luck.
Top 10 should be RecA- just as DH5a is, so that shouldn't be the problem. However sometimes strains do get messed up. I would suggest if it worked fine for you before in DH5a that you go back to that strain and try it instead. You could also try shortening how long you grow the cultures, you might get better results if you harvest the culture soon after it reaches stationary instead of letting it grow overnight.
I would recommend trying stbl3 or NEBstable cells and growing the large cultures up at 25-30 degrees C and see if that helps.
- Badges
- Science topic