NH4Ac-Miniprep becomes bad - why?

Ralf Spenneberg spenneb at uni-muenster.de
Fri Aug 30 08:27:09 EST 1996


In article <503r7p$49m at rrzs3.uni-regensburg.de>,
*@alf1.ngate.uni-regensburg.de (*) wrote:

> Hi there
> 
> Recently I switched from the Qiagen miniprep protocol to an
> alternative method using an RNase containing resuspension buffer as
> well, lysing with NaOH/SDS but precipitating genomic DNA and proteins
> with 6M Ammoniumacetate. Precipitating the supernatant with Isoprop
> gave a yield of plasmid (sequencable) as high as 25ug from a 1.5mL
> culture. 
> So I made a kit containing all the components that I need for the
> prep. After a while (month) though my yields decreased rapidly until
> the method didn't work at all anymore. So I made fresh solutions and
> *bingo* it woked again. Now, after another month, the same problem
> reoccures.
> It would be much easier if I knew *what* solution is becoming bad and
> I would be interested in *why* it happens and how to *avoid* it.
> Anybody with similar problems???
> 
> Thanks
> Mike

Hi Mike,

the only solution in the alkaline lysis protocol i know of that goes bad
is the NaOH/SDS.
The NaOH reacts with the CO2 in the air. This screws up the lysis as well
as the pH after neutralizing with NH4Ac.
One more thing to take care of: Don't allow the SDS to precipitate. Warm
the solution if thats the case!

Ralf.

-- 
Ralf Spenneberg
Klinische Forschergruppe für Endothelzellbiologie
Universitaet Muenster
Von Esmarchstr 56       48149 Muenster        Germany



More information about the Methods mailing list