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-   -   Ammonium or Potassium Acetate? (http://www.molecularstation.com/forum/protocols-methods-forum/18229-ammonium-potassium-acetate.html)

Wolfgang Schechinger 02-03-2006 11:43 PM

Ammonium or Potassium Acetate?
 
Dear experts,

some miniprep protocols use ammonium acetate instead of potassium
acetate. Is there any advantage or is this just another variation of
the theme?

Best,

Wolfgang

Please reply to the group, the email ends in auto trash


J Winkler 02-04-2006 01:18 AM

Ammonium or Potassium Acetate?
 
Wolfgang Schechinger wrote:

Ammonium acetate (typically at a final concentration of 2.5 M, e.g.
Morelle (1980) in Focus 11(1)) supposedly achieves better deproteination
of the lysate.

However, the ammonium acetate solutions are not particularly stable &
most commercial kits would seem to use sodium or potassium actetate.

JW (definitely not an expert)

J Winkler 02-04-2006 01:25 AM

Ammonium or Potassium Acetate?
 
J Winkler wrote:
Sorry, the year for this one must read 1989, not 1980. Regarding
deproteination, it refers to Crouse & Amourese (1987), Focus 9(2), p.3.
Both Focus issues are available for download from Invitrogen, btw.

DK 02-04-2006 04:47 PM

Ammonium or Potassium Acetate?
 
In article <1139060217.934288.190800@g47g2000cwa.googlegroups .com>, "Wolfgang Schechinger" <[Only registered and activated users can see links. Click Here To Register...]> wrote:

That's because you made a mistake. AmAc is supposed to be added
to a final 2-2.5M before precipitation with ethanol (e.g., 1/3 volume
of 7.5M vs 1/10 volume of 3.0M for KAc). BTW, K+ is definitely better
than Na+ for minipreps because KDS is so much less soluble than NaDS.

Total ionic strenght is much higher leading to a better deproteinization.

DK

Dr. Hiranya S. Roychowdhury 02-04-2006 06:36 PM

Ammonium or Potassium Acetate?
 
The both Amm-Ac and K-Ac does do the job fine. Ammonium is to be avoided if
you are using the DNA for ligation, since Ammonium ions seem to interfere with
the reaction.

The reason KOAc produces a big "precipitate" from the alk lysis buffers is the
fact that potassium salt of dodecylsulfate is insoluble in water. When you
add KOAc to ppt the NA, the Na-DS is converted to K-DS.

Quoting Wolfgang Schechinger <[Only registered and activated users can see links. Click Here To Register...]>:



--
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Dept. of Health and Public Services
Dona Ana Branch Community College
Rm# 191T,
New Mexico State University
Las Cruces, NM 88003


Duncan Clark 02-06-2006 09:08 AM

Ammonium or Potassium Acetate?
 
Historians believe that in newspost
<[Only registered and activated users can see links. Click Here To Register...].ne t> on Sat, 4 Feb 2006,
Dr. Hiranya S. Roychowdhury <[Only registered and activated users can see links. Click Here To Register...]> penned the following
literary masterpiece:

I don't know about it's inhibition in ligation but ammonium ions should
be avoided in EtOH pptations if subsequently using T4PNK.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

J Winkler 02-06-2006 11:27 AM

Ammonium or Potassium Acetate?
 
Wolfgang Schechinger wrote:

Wolfgang,

here is the link to the Focus archive:
[Only registered and activated users can see links. Click Here To Register...] .

Regarding your other question: sorry if I did not word it correctly - a
final concentration of 2.5M Ammonium Acetate will form a precipitate
with an alkaline lysate. The cleared lysate is then isopropanol
precipitated. A protocol is available in the Morelle article in
[Only registered and activated users can see links. Click Here To Register...]

In my hands, this protocol consistently gives high quality, stable
miniprep DNA. I learnt of this protocol a couple of years ago here on
this newsgroup, btw :).

Johannes

Dr. Hiranya S. Roychowdhury 02-06-2006 08:45 PM

Ammonium or Potassium Acetate?
 
You are right ... it is the kinasing reaction, not the ligation reaction.



Quoting Duncan Clark <[Only registered and activated users can see links. Click Here To Register...].com>:



--
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Dept. of Health and Public Services
Dona Ana Branch Community College
Rm# 191T,
New Mexico State University
Las Cruces, NM 88003


Bean Long 02-08-2006 03:01 AM

Ammonium or Potassium Acetate?
 
Wolfgang Schechinger wrote:

AmAc will be completely volatile whereas the K salt is not. Very handy
if you plan to concentrate by evaporative methods.

--
Bean

Remove "yourfinger" before replying

dda 12-19-2012 05:29 AM

Re: Ammonium or Potassium Acetate?
 
i think both done the same job, KAc provides neutral environment to form ds plasmid DNA after alkaline lysis and form a network with SDS which help to ppt genomic DNA and proteins. So supernantant contain only re-annealed plasmid DNA which is ppted by ethonol afterwards.
but i read that NH4Ac specifically use to recover large DNA but sorry i also don't no the reason. be careful NH4AC inhibit T4PNK.


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