precipitation of oligos
Anand K. Bachhawat
1) What is the best method to get 17-mer and 20-mer oligos in aqueous
solution (that have been used for sequencing) back into a powder form?
2) What kind of recovery does one get when one precipitates 17-mer and
20-mer oligos using (a) ethanol and (b) isopropanol ? Are there any
carriers that would help in getting a better recovery?
Thanks in advance
Anand Bachhawat
Andre Hamel
If the oligos are in water have you considered simply vacuum drying in a speed
vac or lyophilizer (sample is first frozen at -70oC then kept frozen by virtue
of COLD trap (for example dry ice/ethanol) attached to vacuum line in
between sample container and vacuum pump.
Unless such oligos are quite concentrated in first place, they likely
won't precipitate very efficiently in presence of alcohols (eth. or isoprop.)
Andre Hamel
Manitoba Vet. Virol.
Winnipeg, Manitoba
Canada
email: ha...@ccu.umanitoba.ca
the End
I can suggest what not to do. I routinely rid my PCR product of oligos
by a simple isopropanol precipitation after bringing up to 2 M ammonium
acetate. (Eaual sample volume alcohol). With labeld oligo's, I get
background levels of signal in the precipitate.
I would look for somthing in a sodium acetate ethanol precip. and test it
with labeled samples.
Jim
J. Graham
cha...@wehi.edu.au
There is a paper (Paithankar & Prasad, (1991) Nucleic Acid Rearch 19(6):1346)
you may refer to. They showed that using 2 vol. of ethanal and 10mM MgCl2 at RT
(20-22 degree C.) for 10min, at least 26mer fragment (pUC13 DNA digested by
HindIII) was precipited and higher recovery of low concentration DNA than
precipitation at 0 degree C. was obtained.
Please note that the fragments in the paper were dsDNA, while your
oligos are single stranded.
Good Luck.
Zhonglin Chai
Melbourne
cha...@wehi.edu.au
There is a paper you may refer to: Paithankar & Prasad (1991) Precipitation of
DNA by polyethylene glycol and ethanol. Nucleic Acid Research 19(6):1346.
They precipitate the HindIII-digested pUC13 DNA using 2 vol. ethanol
and 10mM MgCl2 at room temperature for 10min and the smallest fragment of the
digestion (26mer) was precipitated.
Please note that the fragment in the paper were dsDNA while your oligos
Ernest Retzel (1535 49118)
with 80% acetic acid, and then extract the trityls and acetic acid with
5 volumes of ether, and spin in a microfuge. The pellet is washed 2x
with ether and re-spun each time, followed by drying in a speedvac.
I have never tried it without the acetic acid, but suspect it would work.
In the protocol above, oligos ppt quite quantitatively. It lacks the art
of assorted salts and alcohols; it just works.
If the oligo is just in water, speedvacing to dryness works just fine.
Ernie Retzel
ern...@lenti.med.umn.edu
Cornelius Krasel
might be confused...
Current Protocols suggests precipitatioon with 6 vols acetone for
oligos. They claim it to have better effect than the usual ethanol
precipitation. I didn't try it, though.
Cornelius.
--
/* Cornelius Krasel, Department of Physiological Chemistry, U Tuebingen */
/* email: kra...@studserv.zdv.uni-tuebingen.de */
/* "People are DNA's way of making more DNA." (R. Dawkins / anonymous) */
ru...@embl-heidelberg.de
Hi everybody,
a) why does nobody mention the easiest and fastest way, without any
salts beeing involved : the n-butanol precipitation of oligos ??
You simply take 1 part oligo solution, add 9 parts n-butanol and vortex
it vigorously to homogenize the two liquid phases. Afterwards you spin the
precipitate down using a tabletop centrifuge (15 minutes at high speed)
and discard the supernatant -- that's it !
!This even works straight from the ammonia solution!
The method was described by Sawadogo and VanDyke in NAR, Vol19, 3, p674
and is used by us in EMBL regulary for our in house synthesis service.
b) why do people still use PAA gels to check the purity of an oligo and
NOT the much faster and less time consumtive way by running the oligos
on a thin layer chromatography plate??
Take a silca gel 60 F254 plate (0.2mm) on aluminium (Merck), apply your
sample in 2 microliters of H2O and run the tlc plate in a tlc chamber
for 45 min using the following run buffer:
n-propanol 55% v/v
conc. ammonia solution 35% v/v
water 10% v/v
This method is very sensitive (down to app. 50pmol of oligo will be seen)
and was described by Wu et al in "oligonucleotide synthesis: a practical
approach" IRL press, some years ago :-)).
It is used regulary in our lab as well !!!
Hope this helps,
Thomas RUPP
DAVID F. BISHOP
Thomas RUPP writes:
>a) why does nobody mention the easiest and fastest way, without any
>salts beeing involved : the n-butanol precipitation of oligos ??
>You simply take 1 part oligo solution, add 9 parts n-butanol and vortex
>it vigorously to homogenize the two liquid phases. Afterwards you spin the
>precipitate down using a tabletop centrifuge (15 minutes at high speed)
>and discard the supernatant -- that's it !
I tried to get our group to use this method but got resistance because of the
volumes involved. Our synthesizer produces its 0.2 micromole of oligo
in a volume of about 2 ml which would generate 15-20 microfuge tubes and
the hassel of recombining the precipitated oligo fractions.
Individuals ofted have 3-6 oligos to work up and this method seemed too
much trouble.
However, I would dearly love to save our vacuum pumps from all this ammonia
vapor. Does anyone have experience with using smaller volumes of butanol
or with centrifugation at lower speeds (but not for 2 hr)?
David Bishop | EMail:Internet:Bis...@msvax.mssm.edu
Div. of Medical and Molecular Genetics | Bitnet:Bishop@msrcvax
Mount Sinai School of Medicine, Box 1203 | Phone: (212) 241-6946
Fifth Avenue and 100th Street | FAX: (212) 360-1809
New York, NY 10029 |
ru...@embl-heidelberg.de
>
> Thomas RUPP writes:
>
>>a) why does nobody mention the easiest and fastest way, without any
>>salts beeing involved : the n-butanol precipitation of oligos ??
>
> vapor. Does anyone have experience with using smaller volumes of butanol
> or with centrifugation at lower speeds (but not for 2 hr)?
>
> David Bishop | EMail:Internet:Bis...@msvax.mssm.edu
Hi David,
we also using an up-scale of this method in our lab:
Add 25ml n-butanol to 2ml ammonia solution in an 50ml Falcon tube (or
equivalent) and spin at about 5000 rpm in a suitable centrifuge for
about 20 min.
If you use smaller volumes of n-butanol the two phases won't homogenize
properly during the vortex step!!
Or spin it down in a Sorvall centrifuge (the one used for large scale plasmid
preps) using a SS34 rotor with the 36ml tubes for 20 min at 20000 rpm!
ThomasRupp
Jouko Kalevi Pettersson
> However, I would dearly love to save our vacuum pumps from all this ammonia
> vapor. Does anyone have experience with using smaller volumes of butanol
> or with centrifugation at lower speeds (but not for 2 hr)?
I scaled the method up. I use 15 ml screw cap tubes and spin them
for 10 min at 2000 G. For the cleavage of oligos from the CPG cartridge
I use 1.5 ml of 25% ammonia. This amount goes into 10 ml of the
n-butanol phase completely. The yield is usually more than 90%.
I dissolve the precipitate into 1 ml of ultrapure water and repeat the
n-butanol precipitation and dry in Hetovac. No troubles with ammonia
vapour anymore!
--
Jouko Pettersson Email: Jouko.Pe...@Helsinki.Fi
Department of Biochemistry
University of Helsinki