Problem with maxiprep
Posted 16 July 2011 - 07:15 PM
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
Posted 16 July 2011 - 07:32 PM
Edited by sciencelover, 16 July 2011 - 07:34 PM.
Posted 17 July 2011 - 10:45 PM
so I normally try to use multiple midi