I am trying to do a routine maxiprep of a plasmid for AAV production.
The plasmid is pAd Delta F6 (AAV helper virus from UPENN). I 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.
I used this overnight culture to inoculate a larger volume of 600ml for maxi prep.The overnight culture was grown in LB+Amp(100 ug/ml) at 37C
I got final volume of 200ul with a conc of 133ng per ul....
This is the best conc I have acheived in 4 attempts that I made to perform maxi prep. There is probably no technical issue because when I perform maxi prep for other plasmids simultaneously with the same solutions and set up they give good yields as expected.
Any suggestions on how to improve the yield??
I would like to suggest you to use 250-300 ml LB medium for the maxi-prep and harvest 16-18 hours old culture for palsmid isolation. I am sure this would help to achieve about 1000ng/ul plasmid DNA.
@ Tamer - I guess it is better to use Stbl3 specifically because it is known to be more 'stable' for cloning of viral constructs as it avoids ITR recombination events. Do you have some other suggestions in this context?
I haven't tried the CsCl method for DNA isolation.
However, I did try once a midi-prep from 16-18 hours old culture harvested for palsmid isolation which worked well.
Ye Xie, which Maxi prep kit are you using?
I'm having the same low yield problem using the Zymo Maxi Kit reagents.
Thanks
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