Maxiprep woes

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue Oct 24 09:02:13 EST 2000


In article <39F589DA.B9E342ED at immv.unizh.ch>,
  rogerrabbit at rzu-mailhost.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?!
> 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...
>

I used to get odd ball low yields on Qiagen maxicolumns for no apparent
reason, too, and what really used to bug me was I know that the same
plasmid and same bugs used to give me more than a milligram from 50 ml
superbroth after CsCl purification. (Overnight culture, BTW, which is why
I doubt the "bugs chewed up my plasmid" excuse).

FWIW, I've seemd to have better consistency with BRL's "Concert" maxis
(The "high purity" ones, anyway).

Can you save some of the "pre-column" plasmid? Just take a half a ml or
so before the column, phenol extract, EtOH ppt. like you are doing a
miniprep, and run some on gel just to see if you have plasmid (and
estimate the pre-column yield). If you could show that there was plasmid
in the prep before you put it on the column,that would indicate a problem
with the column and not with the prep.

Nick

--
_______________________________________________
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu


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