wierd plasmids: maxi-prep never work - (Aug/04/2004 )

One of my colleagues had a similar problem. The person could never get it done. We all tried to help but never worked. Finally, the vector was changed and then the person could do get some DNA.

Its mysterious sometimes !!!

I've been using the pcDNA3 family of plasmids for over 10 years and have made over 30 different clones for Qiagen maxi, mega, and giga preps, so I have a lot of experience with what you're describing.

When it comes to the viral genes I've cloned, some genes will make the plasmid behave as you've observed. It is definitely due to the particular gene, and I've seen this when the plasmid+insert is much smaller than 10 kb. I work on human and mouse CMV, and we can tell you from our lab that the HCMV promoter/enhancer in the pcDNA3's is active in lab E. coli strains. If you plate a clone with the beta gal gene driven by the HCMV promoter/enhancer (in pCMV-beta, for example), the colonies will be deep blue on X-gal plates. If the viral gene is moderately toxic for E. coli, the colonies with the insert with the correct orientation will be tiny compared to the wrong orientation or vector alone clones. Also, it's much easier to miniprep these toxic clones than to do a large scale prep, as the higher number of doublings in the big volume makes the plasmid vulnerable to loss. So, I'm 99% sure that it's your gene of interest affecting the bacteria, and that it's not the Qiagen column or the purification.

So, when I run into this problem, I essentially do what xysun said, with: use a colony from a transformation fresh that day (even a day or 2 at 4oC can be detrimental), grow it in 2 to 10 ml LB/amp all day, inoculate the whole into the megaculture and grow overnight). In a few cases, I've found that a lot of large scale prepping problems can be avoided when the bugs are initially transformed using a limiting amount of the pure plasmid (0.1 or 1 pg) instead of 1 ng or higher. In addition, I ALWAYS "phenol screen" or miniprep 1 ml of the maxiculture to confirm that the plasmid is in the bacteria, it's intact and not recombined, and that the total mass of plasmid in the culture is worth Qiagen prepping (EndoFree giga preps are expensive.) I've also reduced the amp concentration in the liquid culture down to 50 or 25 ug/ml, and that can help. I never found growing the bugs at 30 oC to help. Finally, I've found in 2 cases that I could never clone the gene into pcDNA3 plasmids, but I could clone them and express them in a similar plamid that has a 5' intron (pCMV's for example.) Presumably, the intron in the E. coli throws off the reading frame for your toxic gene or adds a N-terminal leader that decreases the genes toxicity.

Good luck. I have a lot of explanations as to why each of these tricks work, but it's ultimately voodoo without rigorous testing.

THANKS A LOT CHRISMO for all these explanations!

I will follow all your recommandations this week... And post my results on the forum!

I am interested in your voodoo explanations...

I never let any news about this problem but I am back.

I followed all chrismo's advices and the maxiprep finally worked!

Thanks to everybody...