Dear Members,
 
My problem is, that during the centrifugation before m.i. I often precipitate
my DNA.
 
My conditons:
- 50000 rpm's in Tl100 @ 4 centigrades
- 200 ng DNA per microliter in the m.i.buffer (ca 0.05 M phosphate buffer at
pH7.2)
 
Most of my DNA is made either by the Wizard Maxiprep kit from Promega or the
Qiagen kit. Usually it is eluted in water and stored at -20 centigrades in
aliquots.
The first time an aliquot is thawed, it usually works, when it is reused, it
suddenly stops working.
This looks like a gradual aggregation of the DNA. When the aggregates become
too big, they are simply precipitated.
 
Is CsCl purified DNA behaving better?
Is it worthwile storing the DNA in TE at 4 centigrades and precipitate an
aliquot before each use?
Is my centrifugation speed too high? Much lower often results in serious needle
clogging.
Is filtration a useful alternative to centrifugation?
 
I think most helpful would be a set of conditions, which routinely work for
you.
 
Thank you very much, any hint is appreciated!