isoamyl alcohol

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Tue Mar 27 23:01:04 EST 2001


OK ... call me a heretic -- and my comments blasphemous -- but there are a
few "factual errors" in both the editions of "Maniatis". Amazingly, some of
these are carried over from the first and into the second edition.

I am not sure of the NaOAc - SDS observation. SDS is soluble and PDS is not
... this being the principle that led to the choice of KOAc over NaOAC in
the classical alkaline lysis. Few Grad students (and even some beyond) ever
actively think about this not so subtle implication. I use KOAC for
everything and  sometimes I use AmmOAC. I used to use NaOAC several years
back (as a Grad student) and have also used NaCl (Primarily for RNA work),
but have never observed the phenomenon of NaOAC ppting. My Initial reaction
("Eh!") was because of that. 

Now, about the observation re: SDS-NaAc fluffy stuff: I believe that SDS
pptd out due to local conc. increase. If you add 10 or 20% SDS directly to
2M NaOH while making a fresh bactrial lysis solution, same thing would be
observed. To make the solution, one should add the reagents to water, or
water to one of the reagents before adding the second. NaCl, having a far
greater dielectric point over its acetate counterpart, will have little
difficulty going back into solution. I doubt, therefore, that the fluffy
ppt is a result of EtOH or 2-propanol, per se. 

BTW, I am at a loss about the 50ng/uL DNA in 10% SDS. I have to again do
the "Eh!" for that. :-]

Kornelius' "tight precipitate" (with KCl and SDS) observation is explained
above (SDS v PDS). This is independent of the conc. of either of the reagents.

I am suffering from flu presently ... so if I made any mistakes in the
explanations, somebody, I am sure will be gracious enough to correct.

Hiranya


At 11:49 PM 3/26/01 +0200, Gys de Jongh wrote:
>"David F. Spencer" <dspencer at is.dal.ca> wrote in message
>news:dspencer-F875B7.17361622032001 at News.Dal.CA...
>> In article <3.0.6.32.20010318081653.007bcb30 at cnmailsvr.nmsu.edu>,
>> hroychow at nmsu.edu ("Dr. Hiranya S. Roychowdhury") wrote:
>>
>> > At 02:54 PM 3/18/01 +0100, Gys de Jongh wrote:
>> >
>> > >
>> > >Hi,
>> > >if you use sodium acetate the SDS will precipitate.
>> > >The SDS stays in solution at high NaCl concentrations.
>> > >See maniatis appendix
>> > >--
>> > >Gys
>
>Hi,
>got the flu , been away for a few days.
>Here is the reference :
>
>1) Molecular cloning
>    Sambrook,J
>    second edition 1989
>    Volume 3
>    Appendix E : Commonly Used Techniques in Molecular Cloning
>    subject : concentrating nucleic acids
>    subtitle : precipitation with ethanol or isopropanol
>    page : E.10
>
>quote : Sodium chloride (0.2M) should be used if the DNA sample contains SDS.
>The detergent then remains soluble in 70% ethanol.  Sodium acetate
(0.3M;pH 5.2)
>is used for most routine precipitations of DNA and RNA. unquote.
>
>[ See the problem in the original post ]
>[ Subject: isoamyl alcohol ]
>[ From: Ron Kim (rkim02 at emerald.tufts.edu) ]
>[ Date: Wed 07 Mar 2001 - 16:31:44 GMT ]
>
>
>Some time ago a had a solution of 10% SDS containing about 50 nanograms per
>microliter DNA . This was standing on my lab table in a clear tube. I
wanted the
>DNA. I started by adding NaAc/HAc (3 M pH 5.2 ref 1 , page B.13). The tube
>filled itself immediately with a large fluffy white cloud. One of those
days I
>thought , started reading and found ref 1. I repeated the experiment .
This time
>by adding NaCl 5M , final concentration 0.2M (ref1 table E.1 page E.11) In
about
>half of the tubes I saw a faint precipitation which went back in solution
apon
>gentle agitation. Support for the observation in ref 1 I think (?) I then
added
>2 vols of ethanol , cooled etc and finally got the expected small pellet
which
>proved to be rather clean DNA in subsequent experiments.
>
>[Thought of Hiranya ]
>[ Subject: Re: isoamyl alcohol ]
>[ From: Dr. Hiranya S. Roychowdhury (hroychow at nmsu.edu) ]
>[ Date: Sat 17 Mar 2001 - 13:19:09 GMT ]
>[ With acetate you'll get unsoluble sodium acetate. ]
>[ Hmmmmmmm! Interesting observation ... ]
>
>> >
>> >
>> > Eh?
>> > Dr. Hiranya S. Roychowdhury
>> That sums up my reaction as well!
>>
>> I did a brief look through Sambrook et al. but couldn't find such a
>> statement. Someone must be confused.
>>
>> Dave
>
>This effect amazes me too . I think there may be confusing about what is
>precipitating. I think we may safely conclude that this must be SDS and not
>sodium acetate. There is no further ref or explanation in ref 1.
>Annyone ??
>
>[ Suggestion of Cornelius ]
>[ Subject: Re: isoamyl alcohol ]
>[ From: Cornelius Krasel (krasel at wpxx02.toxi.uni-wuerzburg.de) ]
>[ Date: Thu 22 Mar 2001 - 08:49:25 GMT ]
>
>I tried his suggestion today.
>I added KCl 4M , final concentration 0.2M , to a 10% solution of SDS. This
>resulted in a very solid precipitation. Repeated it with 5% and 1% SDS
with the
>same result. Seams Cornelius found a nice alternative solution of the
problem.
>
>--
>Gys
>If all else fails , read the directions :)
>
>
>
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu

---




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