Hi,
I am trying to optimize plasmid yield to prepare for animal experiments. So far, I can only get about 0.6~0.7mg from 1L starting culture.
I used the Zymo Max prep kit, and I don't think that I over-lysed/under-lysed the cells during isolation. My vector is pcDNA3.1, which is high-copy-number, and the plasmid was transformed into DH5alpha cells (also tried two other strains, this strain gave slightly higher yield). I've tried Terrific broth vs. LB or 2xYT, and I optimized the OD600 to 3~3.5 before I isolated it. I also tried to add chloramphenicol to my culture media and started with a single fresh colony.
I am wondering if there is any way to optimize the backbond to increase the yield, like adding or altering some gene features or elements? OR any other advice on increasing plasmid yields. Thank you!
0.7 mg is not so bad. pcDNA3.1 is quite large, 5.4kb and the smaller the better. However, depending on what you want to do with it, you might want to keep all of it. I would have expected that Terrific broth would do better. Increased oxygenation (shaking) and antibiotic concentration might help.
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