Low yields with quiagen midi kit - (Jan/17/2012 )
I´m having some problems with quiagen midiprep kit.
I´m using pQEβG (pQE beta giardin) plasmid (low copy plasmid) and I get very low yields, for example 6.25 ug (using 50 of buffer TE)
I´m using the following protocol:
- Pick a 5 colonies from a freshly LB agar plate with ampicilin and kanamycin and inoculate a starter culture of 10 ml LB medium containing ampicillin and kanamycin; incubate for approx. 24 h at 37°C with vigorous shaking (approx. 120 rpm).
- Next morning, inoculate 1 mL of starter culture with 2x 100 mL LB medium with ampicillin and kanamycin and divide in 2 flasks; incubate 24 h at 37°C with vigorous shaking (approx. 120 rpm)
- Next day I make DNA extration using QIAGEN Plasmid Midi Kit
- I´m using 2 quiagen tip 100
- I use 4000 r.pm. for all centrifugations (maximum speed is 5000 r.p.m with that rotor) and the times are the ones in the quiagen midi prep protocol
- I redissolve the DNA in 50 or 100 µl of TE buffer.
- Possible causes: isopropanol; low speed centrifugations; starter culture steps
Try using the whole 200 ml in the prep, it should work fine. It is also possible that you are not maximising the amount of plasmid being produced - the overnight culture in your 100 ml should only be 16-18 hours, longer than that and the culture reaches a density at which the plasmids are not replicated efficiently or something like that (can"t recall at the moment).
If your strain is not chloramphenicol resistant, you could also try chloramphenicol amplification of the plasmid: