Problem with maxiprep - (Jul/16/2011 )

Hi all,

I'm using Nucleobond kit to extract my plasmid DNA and recently I encountered problems to purify a retrovirus backbone plasmid (around 12kb) from maxiprep. I had no problems with bacterial transformation and miniprep. After I inoculated miniprep bacteria into big flask for large prep, bacteria grew well, every step looked fine but when I run DNA gel I found that there was only one band for uncut plasmid (looked like being linearized, but i did not add in any enzyme!) and there was smear in digested plasmid lane. To test whether my maxiprep kit is working or not, I have done maxiprep for another plasmid (smaller, around 6kb) concurrently and it was alright for digestion of this plasmid after maxiprep, which means nothing went wrong in my maxiprep. I cannot figure out what the problem is. I used same antibiotics (ampicilin), same LB, same maxiprep kit to prepare both constructs but I failed to produce the larger, lower yield plasmid. It is important to highlight that I have ever used the same kit to produce the retrovirus backbone plasmid for 3-4 times, I just encountered the problem of maxiprep in recent one month. So what makes my large prep plasmid being linearized/degraded during maxiprep? I have tried to pick more colonies from my amp plate and cultured with LB in small volume for miniprep and all clones are positive. I cannot understand why it cannot be purified with maxiprep kit? I will try to re-start from bacteria transformation step and use competent DH5a E.coli prepared by other post-doc in my lab. Anyway I hope I can solve this mystery asap with helps from you all !!! Thanks in advance

charlene

Try using this Zyppy Maxiprep kit. The Qiagen one wasn't working for me (I'm not sure what brand you are using) so I tried this one and it worked. Plus it has colored buffers so you can see if there is complete bacterial cell lysis and neutralization.

Qiagen Maxi sometime does not work for me either,
so I normally try to use multiple midi