Ian A. York
iayork at panix.com
Tue Oct 24 10:18:23 EST 2000
In article <39F589DA.B9E342ED at immv.unizh.ch>,
Susanne Rohrer <rogerrabbit at rzu-mailhost.unizh.ch> wrote:
>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
You're not alone; intermittent low yields from maxipreps is a chronic
problem in virtually every lab I've talked to. Qiagen columns are the
usual culprit, though to be fair they're also the most widely used.
I haven't had a problem with this for a long time now (knock wood), but I
don't know if it's something I've done or if I'm merely lucky--that's the
problem with intermittent problems.
Some things that are worth trying--none of which are guarnateted, or even
necessarily real concerns; there's a little superstition about this:
(1) Use LB rather than TB. For me, at least, it's more predictable. For
high-copy plasmids (e.g. pUC and derivatives) try 100 ml overnight
culture; for low-copy plasmids, try 200 or 250 ml.
(2) Don't use the QiaFilter system. I think that works well when there's
just the right amount of bugs, but it seemed much less consistent to
me. I use the centrifuge to spin out the crud.
(3) Try hard to avoid getting any little pieces of crud into the
column. Usually after a spin there's a good solid pellet, with a few
little flakes floating on top. I pour this into the column through a
piece of gauze, which filters out the last little pieces.
(4) If the solution you have after the spin is not clear--if it's cloudy
at all--pour it right back out of the column. I usually run this through
the Qiafilter, just as a final cleanup.
(5) Make sure you're recovering all the DNA from the final isopronanol
spin. At least half of it often sticks quite high up on the tube and can
be missed if you're only looking for a pellet.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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