insoluble DNA pellet after Qiagen Tip20
bernard at elsie.nci.nih.gov
Wed Jun 22 23:17:02 EST 1994
In article <2uafta$8fv at ysics.physics.sunysb.edu>, mhollowa at epo.som.sunysb.edu writes:
> In article <-210694111348 at dent-226-97.dentistry.umich.edu> @umich.edu ( ) writes:
> >In article <eggermon-200694162025 at 188.8.131.52>,
> >eggermon at popserv.kuleuven.ac.be (Jan Eggermont) wrote:
> >> but the final pellet was as
> >> resistant to resuspension as the first one (despair....).
> >Sounds to me like you overloaded the column with too much bacteria and/or
> >didn't wash enough of the protein (it is your insoluble material)
> I've found that I often get a small amount of column material
> washed out with the eluent. I don't recall seeing any warning
> about this in their liturature. If not spun out, it will
> eventually clump and precipitate of its own accord. It doesn't
> seem to have any effect on the use of plasmid in transfection.
> Mike Holloway
> mhollowa at epo.som.sunysb.edu
I think this problem is not simply limited to the tips that you
describe. I've had the same problem with Stratagene's PrimeErase Quik
push columns (maybe due to some over-zealous syringe work on my part :-) ).
I also traced an anomalous A260 vs DNA-on-gel result to a small amount of
Promega Magic/Wizard material that escaped the spin column (surprisingly,
the A260/A280 ratio was still good). However, this was only a problem when
I was using it for small amounts of DNA (eg. insert from gel). I, too,
can't say that the unwanted material has interfered with any susequent
manipulation but I also favour a quick microcentrifuge spin after using
such adsorption methods.
Keep on purifyin'!
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
Ireland 1 : Italy 0
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