wierd plasmids: maxi-prep never work - (Aug/04/2004 )
I'm having this strange problem with four pcDNA3.1 derived plasmids, each about 10kb in size. It worked well in mini-prep, though I did not get as much DNA compared to other plasmids. But when I did maxi-prep, i got nothing, NO DNA AT ALL?? I repeated twice and did midi-preps, all the same, NO DNA!!! And this only happened to those four plasmids, not other plasimids, so I know there is nothing wrong with the maxi-prep kit. Also, doing maxi-prep I saw nothing unusual, there were lots of bacteria from overnight culture in presence of ampiciline. All four plasmids contains the DNA encoding the same gene of my interest, so I'm thinking maybe that gene somehow affects bacteria......??? Anybody had similar experience??? Any suggestions pleaseeeee!!!
Do your maxipreps have plasmid size specifications? Could they be different to those for mini-preps?
I had met the same question when I was purifing a plasmid, the size of which is more than 20 Kb.
In my experiment, it seems that the long-time culture(more than 20 hours) or high-speed shaking(300 rpm) is dangerous for the large-size plasmid in the maxi-pre.
So I solved this question as doing like this: in the morning of the first day, pick a clone from a freshly-streaked plate, culture in 10 ml LB about 8 hours, then make a maxi-culture with 1:500~1:1000 concentration for 14~16 hours, the shaking speed is 200~250 rpm. from 800 ml cultural medium, I got 4 mg plasmid this time.
Thanks all for the replies.
Fist of all, my plasmids are only 10kb in size, and I have very good yield of my many other 10kb plasmids. Only these plasmids with this specific gene in it are giving me big problems! Does anyone has similar experience???
2 years after you I have the exact same problem: a pcDNA3 plasmid oh 10 kb that gives DNA in miniprep but not in maxiprep!!! Does anyone know how to help me?
I don’t think the gene could affect bacteria, because it couldn’t express because pcDNA3.1 has CMV promoter for expression of genes, and that promoter is active just in Eukaryotic cells, not in bacteria.
And, if you do minipreps without problem, maxipreps should work without problem too!!! The plasmid IS definitively in the bacteria!!!
Some kits works well just with small plasmid (less than 10 kb) and/or your efficiency decreaces when size’s plasmid increases.
What kit are you using??
May be give the old, time consuming, but more relieble CsCl method a try?
on the very rare occasion that this has happened to me, there was a problem with the media. For ex, perhaps antibiotic dilution was wrong? perhaps LB was made incorrectly, or carmelized during autoclaving? perhaps that batch of flasks did not get the soap rinsed off them when they were last washed?
anyways, if you haven't yet, try to re-do one more time with entirely new LB, with careful attention to everything, and see if it works? sorry if this isn't helpful...
Thanks for reply...
I tried 3 different times with 3 different LB + amp media, and it never worked. In parallel I prepared other pcDNA3 plasmids < 10 kb and it worked! I use qiagen maxi kit and I tried both 100 mL and 500 mL culture (20h).
Maybe I should indeed try other kits. Let me know if you have other ideas...
I keep in touch...
have you called Qiagen's tech support?
I'm thinking that it's crazy to change kits unless you're sure that's the problem, that gets expensive quick