Problems with plasmid DNA extraction - (Jul/14/2013 )

Hi guys!

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?

Thank you!!

I don't quite understand the use of chloramphenicol since these appear to be kan and amp resistant plasmids, but I will trust you know what you're doing.

I had a similar problem which was solved by growing at a lower temperature. In my case, the cultures would never grow to turbidity at 37C.

I read that plasmid DNA yields can be improved by adding chloramphenicol (170ug/ml) to the culture medium (Molecular Cloning, Sambrook and Russel), but in this case it didn't work.

I'll try lower temperatures! Thank you!

Hi there!

Lower temperature didn't work!  

Actually, the culture growth was fine but after doing a midiprep, I got only 200ng/ul of plasmids (with RNA contamination)!  I need, at least, 500ng/ul for a transfection protocol. By the way, can I use DNA simple with RNA contamination for transfecting mammalian cells?

Do you, guys, think that the problem is the XL1-blue? Should I try another strain, like DH5 alpha? Should I try something else? 

Any suggestions? 

Thank you very much!