Maxiprep woes

[Cornelius Krasel]

Susanne Rohrer <"srohrer(removethis)"@immv.unizh.ch> wrote:
> 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
> options?!

1) Use a high-copy plasmid. With pcDNA3-based plasmids, we get more than
   1 mg of DNA out of a Qiagen maxi column from a 250 ml culture which
   is probably the capacity limit of the column. The culture volume could
   probably be reduced much further, but I am too lazy to test this.

2) For Qiagen: do the precipitation step at 4°C over night. For the
   subsequent centrifugation step, use 50 ml Falcon tubes in a swingout
   rotor; we centrifuge at 4500 rpm (approx. 3800 x g) in a Hettich
   Rotanta 96R. This is sufficient to pellet the DNA, and because of
   the swingout rotor, it can be easily collected at the bottom of
   the Falcon tube. (I used to do this with Corex tubes in a SS34
   equivalent before. Switching to the method outlined here I increased
   my yield by at least 50%.)

3) Don't wash with 70% ethanol.

HTH,
--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */