hello - I am working with a mixed baterial population for herbicide bioremediation. I would like to characterize plasmid DNA in my culture by size. I am particularly interested in seeing if I have P.ADP-1plasmid, size around 100kb (very large). what is the best method to conform the presence of whole P.ADP-1 plasmid in the bacterial population.
many thanks!
IF you know the sequence, design primers and do PCR, then electrophoresis. if plasmid is threre, you will see a band on electrophoresis.
PCR sounds like your best, cheapest option, though ideally you'll want to start with a purification step, especially if you have similar sequences in your population but they aren't the same length.
You can use the protocol published by Kado and Liu quite a time ago. It works in a way that plasmids are not sheared and so you can see plasmids of sizes more than 100 kb. Normal column based protocols shear larger plasmids.
For more information have alook here: Common origin of plasmid encoded alpha-hemolysin genes in Escherichia coli.Burgos Y, Beutin L.
BMC Microbiol. 2010 Jul 19;10:193. doi: 10.1186/1471-2180-10-193.
best regards
Lothar Beutin
- Badges
- Science topic