Pleeease, I need your help!
I'm working with pEGFP-C3, pHM6-HA and pDEST-V5 plasmids (with the inserts) in order to use them in cell culture. Theoretically, they are high copy plasmids. Growing 1 single colony in 5 ml of LB media should be enough. But it is not! The last time I use a mini kit (Sigma) for extraction, I got less than 50 ng/ul (20ul) of a impure DNA. Oddly, it happens only with these 3 plasmids. I try the same kit with my pTRG plasmids, and it worked fine (more than 300ng/ul).
Well, I use XL1-blue competent cells for subcloning, grow them in 5 ml (+ atb) at 37*C overnight and extract de DNA. It should be simple! And I normally do the toothpick minipreparation in order to identify bacterial colonies containing the recombinant plasmid (molecular cloning manual).
I already tried doing a Midi prep with alkaline lysis (without kit) and the plasmid's amplification using chloramphenicol... and nothing!
Do you guys think that my plasmids might be toxic for the cells? Should I grow them in 30*C? Any suggestions?
Edited by lschier, 14 July 2013 - 12:30 PM.