hroychow at NMSU.EDU
Fri Sep 8 16:22:31 EST 1995
On 7 Sep 1995, Sergey V. Orlov wrote:
> R. Woodward wrote:
> >Dear All
> >Can anyone tell me how to make 3M KoAc and get the pH down to ~4.8?
> >I ahve tried the method suggested in Maniatis but I can only ever
> >achieve a pH of 5.8 at best, for use in alkaline mini preps this
> >does not appear to have any datremental effects. Maniatis makes
> >a solution which ends up being 3M with respect to K and 5M wrt
> >Ac however the acetate ion concentration must be much higher than
> >this after the solution is pHed. For mini preps this appears ok
> >but I am concerned when I use KoAc in other buffers ie for
> >S1 nuclease treatment. Are enzymes more dependent upon the K or
> >Ac ????
> >Thanks Robert
> >R. Woodward
> >Email rw200 at cus.cam.ac.uk
> Dear R. Woodward,
> Indeed, Maniatis's method for KAc preparation don't allow achieve
> pH lower than 5.5. For S1 nuclease treatment pH level is impotant.
> Try use the more strong acid (i.e. HCl) for titration KAc solution.
> Because NaCl is included in S1 buffer, additional amounts of Cl- ions
> will not prevent S1 nuclease reaction.
> Good luck,
> Serge Orlov
> E-mail: Serge at serge.stud.pu.ru
KoAc solution is also required for other manipulations and Cl ions may
not be desirable in those. I make pH4.8 KoAc by simply dissolving
KoAc in glacial acetic acid, so that that almost no water is added. It
takes a while but works fine. Sometimes, though, the KoAc crystalizes in
the 3M solution, but that is not a big deal since warming to RT is
sufficient to redissolve.
Hiranya S. Roychowdhury
Plant Genetic Engineering Lab.
Box 3GL, NM State Univ.
Las Cruces, NM 88003
Phone: (505) 646-5785
hroychow at nmsu.edu
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