how to isolate huge plasmid DNAfrom Pseudomonas putida - (Aug/02/2001 )
MessageI extracts huge plasmid DNA (8 Kb) by modified alkaline lysis method(Kado and liu) and miniprep Kit(QIAGEN)from Pseudomonas putida.I got three bands which size are 25kb,40kb and more than 40kb respectively. but when I difested them by EcoR I, BamH and Hind III, there are always no any bands. please let me know what wrong with it. thanks your help.
This is typical of very large plasmids. What you have in hand is a nicked product. Combine isolation with a CsCl gradient. You can also use a DEAE column for this.
Well, I'm working with P. putida cosmids (~40 kb inserts), cloned in E. coli, though. I don't have any problems with them. I'm using the same Qiagen kit, or even some fast method which is not gentle at all. After digestion with EcoRI the bands look just nice.Maybe you should use more DNA for digestion, and leave it over night.