Hello House,
Please I am facing some kind of difficulties with my DNA during Maxi Prep. I never got any DNA after the whole process. Nevertheless, I checked using mini prep and I could see the presence of my DNA, using premix. I followed the necessary protocol from Quigen Company, but still yields not result. Please, has anyone ever encountered something like this?
Did you add ethanol to your buffer PE? That's a common mistake.
Without more detail regarding exactly what you did and what you saw at each step, it's hard to help you.
In addition to what Govinda mentioned above, this problem (plasmid extracted in mini preps absent when going for maxi) is sometimes seen with unstable plasmids like some lentiviral vectors. In some cases, it is due to the integration of plasmids to the bacterial genome on long cultivation. What is your vector backbone? If this is one of the problematic large plasmids, using specific recombination deficient strains and shorter cultures could help.
Best, Manoj.
Before changing your bacteria you may want to try optimising your Maxi: Grow 1 colony from a fresh plate during the day (3 ml). Dilute 1:500-1:1000 into 150 ml culture medium. Grow Overnight (do not overgrow) and spin. Use ZymoPURE plasmid maxiprep. It is far better than Q. in terms of yield and convenience. for unstable plasmids using half the concentration of antibiotic and reducing growth temperature may also help.
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