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.
In article <email@example.com .com>, "Wolfgang Schechinger" <[Only registered users see links. ]> 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.
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 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 users see links. ]>:
Hiranya S. Roychowdhury, Ph.D.
Dept. of Health and Public Services
Dona Ana Branch Community College
New Mexico State University
Las Cruces, NM 88003
Historians believe that in newspost
<[Only registered users see links. ].ne t> on Sat, 4 Feb 2006,
Dr. Hiranya S. Roychowdhury <[Only registered users see links. ]> penned the following
I don't know about it's inhibition in ligation but ammonium ions should
be avoided in EtOH pptations if subsequently using T4PNK.
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
here is the link to the Focus archive: [Only registered users see links. ] .
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 users see links. ]
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 .
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.