Maxiprep woes

Student mucineer at iname.com
Tue Oct 31 08:44:15 EST 2000


I have had lots of problem with the Qiagen maxi kit, particularly that the
plasmids I want to purify are low-copy number ones. I did however sort out a
compromising solution that leads to about 200-250 micrograms of DNA from a
360mL culture.

What I do is this:

1) Spin 2x160mLs of bugs in LB separately and treat as separate samples
during resuspension, lysis and netralization and filtering with Qiafilter.

2) Filter the two lots of supernatants into ONE Qiagen column and proceed as
normal

3) Spin for 1 hour, and 30 mins with IPA and 70% EtOH respectively rather
than the recommended 30mins and 15mins.

This may be a costly solution but I can't lose much time optimising the damn
thing.

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
> 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...
>







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