NH4Ac-Miniprep becomes bad - why?

Toumy Guettouche tguettou at newssun
Thu Aug 29 18:01:28 EST 1996



On Thu, 29 Aug 1996, * 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
I had the same problem with our Alkaline-Lysis-Miniprep Method. I found 
that it is best to make up the NaOH/SDS solution fresh before using it. 
If you need to store it make sure the bottle is tightly closed. This is 
by the way mentioned in the  Quiagen Midiprep  manual: " After use the 
bottle containing buffer P2 (NaOH/SDS) should be closed immediately to avoid any 
reaction between the NaOH and the CO2 in the air. If the buffer is left 
open for any length of time, it should be prepared fresh from stock 
solution." 
To precipitate DNA I us 3M KAc, which I store at 4deg C. I do 
not know if your problem might be with your AmmoniumAcetate.
Hope that helps
Good Luck
Toumy





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