Minprep Madness

Emir ekhatipo at NOSPAMmidway.uchicago.edu
Wed Nov 1 12:03:00 EST 2000


Did you check the pH after N3-neutralization - should ne acidic to bind to
the column. If not acidic, add ~1/10 vol. 3M NaAc pH5.0. I would also
recommend washing your cells in STE before resuspending in P1, although this
relates more to better final purity of your prep than binding of plasmid to
the sorbent.
Call techserv if you can. They might tell you more.
Emir


<jayni76 at my-deja.com> wrote in message news:8tpc9m$1h$1 at nnrp1.deja.com...
> Please help?? Suddenly, after having done spin minipreps for a long time,
and
> always getting excellent eluted concentrations ie. 500-1000ng/ul, I am
> getting very low yields?? I have carried out the tests to see where the
DNA
> is going and it seems to be not binding to the spin column, but carrying
> straight through with the rest of the lysate!! I have tried 3 different
> qiagen kits, and a sigma kit, I have changed culture volume 2-5ml and
doubled
> P1, P2 and N3. I have streaked glycerol stocks of plasmids that worked
fine
> 1 month ago and that gave the same low yield, 10-100ng/ul.  I use
differetnt
> strains, JM109, XL1 Blue, Nova blue, DH5a, and they are making no
difference!
> It is making my job very difficult, as midipreps take a lot longer!! But
they
> are always good yields. Why can things change so suddenly??  The only
> difference I can see is that lysis doesn't look too perfect and the
> neutralisation step looks a little too much like egg white, gluupy rather
> than fluffy, and when spun it forms a very compact pellet. Has anybody any
> ideas as I have totally run out!!
>
> Thanks
> Jayne
>
>
> Sent via Deja.com http://www.deja.com/
> Before you buy.







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