I am trying to do a routine midiprep of a plasmid for AAV production. The plasmid is pAd Delta F6 (AAV helper virus from UPENN). I got the plasmid directly from the core facility, transformed into OneShot Stbl3 cells, plated on fresh LB agar plates with ampicillin (100 ug/ml). There were plenty of colonies on the plate the next day. I picked a couple of colonies and inoculated fresh LB broth (5 ml) supplemented with 100 ug/ml ampicillin. The overnight culture grew just fine.
Then, I went to use that as a starter culture for a midiprep. I put 50 ml of fresh LB (the same used for the overnight culture) into a clean, autoclaved 250 ml erlenmeyer flask. I added 100 ug/ml ampicllin (which I keep at 1000x stock). I inoculated the flask with 100 ul of the overnight culture (so 1:500 dilution) and put in the incubator to shake overnight (200 rpm) at 37C.
The next day (13 hours later) there is no growth. Nothing. I've done this a couple of times now and it seems plasmid specific. Just to make sure that it's not something else I'm doing, I grew up another set of Stbl3 that contain an amp-resistant plasmid (AAV-shRNA construct) at the same time, and it grows just fine in the 50 ml prep. So what's the deal? How could something grow in a miniculture but not 50 ml?
It's possible I have some false colonies on the original plate, but I picked 3 colonies, and they all grew in ampicillin-containing 5 ml cultures, so I dont' suspect that I got some random bacteria that had amp resistance. Any help would be greatly appreciated.
This sounds like a pretty bizarre observation, but you describe all the obvious controls. It would be interesting to passage 10 ul of your first overnight culture into 5 ml of fresh LB and see if that grows. If not, it would suggest that something changes over time. Perhaps the plasmid is lost or it becomes toxic. In fact, it might be worth doing a miniprep from the small culture to see if the plasmid is still there. However, if the small culture does grow, their must be something about the larger culture such as aeration that somehow has a specific effect. Also, if the second small culture grows, you could just grow up ten and combine them for a midiprep. Good luck!
Stbl2 and Stbl3 with plasmid vectors containing viral DNA should be grown at 30C. Give it a try.
Thanks for the suggestions. I'll give the lower temp a try. And I'm sequencing the plasmid I do isolate from the overnight cultures to ensure I have the right thing.
Just an update. I asked others and they told me that they used Stbl2 successfully with this plasmid. I did that, and now it works. One possibility is that this plasmid is large (15 kb) and Stbl3, for whatever reason, might have trouble replicating? Not sure. But in any case, this was the fix.
I am using the same plasmid but I am facing a different type of problem.
In my case I had also transformed in Stbl3 and the cells grow fine in overnight cultures (mini, midi and maxi) but the plasmid yield is extremely low !! When I use 600ml of the overnight culture [grown in LB+Amp(100 ug/ml) at 37C ] for a maxi prep I get final volume of 200ul with a conc of 133ng per ul....
Any suggestions on how to improve the yield??
Hi Tanja,
Glad this thread helped. Yeah I was puzzled at first with the Stbl3. Then I asked Penn what they do and they said they used Stbl2. I didn't think there would be that much of a difference. People use Stbl2/3 interchangeably usually. But it grows very well in Stbl2 and I've used the preps several times to make virus. Good luck!
Hi Tanja and Shambhabi,
Do you get a higher yield using stbl2 bacteria? I use stbl3 and they are growing fine, but I always get a low yield (around 200ug per maxiprep). I grow them in 30c for 20hr.
Thanks!
Dear Edita,
Normally, I grow 250ml culture for minimum 17hours at 37C and get yield of ~1.3micrograms per microlitre (final volume of 200ul from a midiprep) . I have only used stbl3 for preparing the plasmids, so cannot comment if Stbl2 gives better yield or not.
Hope this helps!
Hi Shambhabi,
Thanks for your answer. Ok then, it's about the same with the yield I got :)
Regards,
Edita
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