2 replies to this topic

[manicd]

Hello everyone.
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

Thanks
Michael

[bob1]

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).

[phage434]

If your strain is not chloramphenicol resistant, you could also try chloramphenicol amplification of the plasmid:
http://www.qiagen.co...0&MenuItemId=56