Miniprep gives high yield but maxiprep gives no plasmid? - (Jun/22/2014 )
My miniprep gives high yield but maxiprep gives no plasmid? Am I losing my plasmid because it is too large or unstable? How can the bacteria grow without the plasmid?
I am trying to produce stock of an 11kb lentivector in stellar cells (i.e. suitable cells for lenti vector). My miniprep had a pretty good yield - 15ug from just 1.5ml culture. The 1.5ml for the mini came from a 5ml O/N (14hrs) culture started from a well isolated single colony on a plate that was less than a day old. I setup two 100ml maxi cultures - seeded one with 100ul and another with 1ml from the 5ml miniculture. The amplicillin was 100ug/ml in both mini and maxi. Maxi was grown O/N for 14 hrs. Both maxi cultures had good sized pellets but no plasmid yield?
I guess I'll be answering my own question since no one replied and I finally figured out the problem.So hopefully it will help others facing the same problem.
I picked a single colony and cultured it in 5ml LB with 100ug/ml ampicillin for 6hrs at 37C 250 rpm. I took 1ml of this 5ml culture and seeded it into 100ml LB with 100ug/ml ampicillin for 14hrs at 37C 250rpm. Just to check, I did a miniprep of 1.5ml and got good yield as I did before - 15ug. I then did a maxi on the remaining 100ml and got 800ug plasmid.
Problem: Miniprep good yield but maxiprep no yield. The cell density of my 5ml culture was sufficient for a miniprep but too high for seeding the 100ml maxiprep culture.
Solution: Grow the starter culture for about 6hrs instead of 17 hrs then use 1ml or 100ul of this to seed the100ml culture and grow for 14-16hrs.
So, the real problem apparently is that long culture of the inoculum overgrew, and lost the plasmid. This is often caused in ampicillin resistant strains when grown in ampicillin containing medium, Switch to carbenicillin for your ampicillin selection. It's much more stable, and will at least reduce the problem. Longer term, use plasmids with a different resistance. We like kanamycin, chloramphenicol, or tetracycline.