I'm trying to recover a 25 Kb native low-copy no. (10-12 per cell) plasmid from S. aureus, but am unable to get a clean prep that will cut properly. I have used several methods including the CTAB method, Promega minipreps and Qiagen large construct kits, all with and without isopropanol extraction following lysis. I do not have the facilities for CsCl centrifugation.
I think this might be something to do with the debris I can see following lysis, which does not disapper even if i add >500ug lysostaphin per ml of resuspension during the lysis step, and therefore I am currently transducing this plasmid into a more suitable host for extraction. Does anyone have any ideas, tips or seen anything like this before?
plasmid prep from Staph aureus.
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