Arnoud van Vliet
avvliet at knoware.nl
Tue Oct 24 16:30:10 EST 2000
Some people still talk with the reps? Gave up a long time ago, although
recently I actually had one who confessed not to know anything about the
subject I was querying about, but would send me all the info I required :)
Coming to your problem, I can only suggest some things:
- spin the isoprop precicpitation in microcentrifuge tubes at full speed.
What I usually did with the midiprep: 5 ml eluate, add 4 ml isopropanol,
mix, fill 3 eps with 1.5 ml mix, spin 15 min full speed, decant supernatant,
add remaining mix to the three tubes, spin 30 min, etc. Works much better
than spinning in the big (usually dirty) tubes.
- I always used 100 ml LB overnight culture (pUC plasmids) per midi column.
Worked fine. The comments about that in all the Qiagen booklets are pure
rubbish! We know use the Qiaprep spin columns, and there you should use 3 ml
culture. We use up to 10 ml without problems.
- Keep as far away as you can from the &%$# QiaFilter. Ritually burn them
(together with the dreadful QiaEx II kit)
hope this helps
"Susanne Rohrer" <"srohrer(removethis)"@immv.unizh.ch> wrote in message
news:39F589DA.B9E342ED at immv.unizh.ch...
> For the n-th time I have done a Plasmid prep and got a really low yield.
> I really hate them. We have tried qiagen, macherey-nagel, both are the
> same. What bugs me is how expensive they are. This time I even wasted a
> maxi column.
> So I've talked to the rep before. First he said to lower the culture
> volume (16 instead of 30 ml for a midi), then we find out you're not
> supposed to use an overnight culture because the cells are already
> starving and chewing up their own plasmids. on the other hand, they tell
> you no, you can't use TB.
> It just seems to me ages ago they used to work fine. I used to get like
> 300 ug from a maxi. What (the =*%+) is going on, and what are my
> I mean, I get higher yields from a miniprep! Why can't they make their
> hydrophobic matrix into maxi columns?!
> Susanne in a state of agony...
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