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solutions to storage oligonucleotides

solutions to storage oligonucleotides - Protocols and Methods Forum

solutions to storage oligonucleotides - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 11-01-2004, 03:08 PM
fabio
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Default solutions to storage oligonucleotides



Usualy I stored primers in water (sterilezed or depc treated if for RT)
at -20 °C;
but i'm reading (on the tecnical scheet of Invitrogen) that the best
solutions for woring stock is TE pH 8(I supose that the EDTA
interfirence with Mg++ isn't so important like I thought).
So I want know your opinion and methods to the best way to store the 100
mM and working solutions of primers.
Someone prefer to store primers dried, but it's not very pratics.
Thanks in advace.

Fabio
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  #2  
Old 11-01-2004, 03:23 PM
Tom Knight
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Default solutions to storage oligonucleotides

fabio <[Only registered users see links. ]> writes:

I store my primers in TE. The amount of primer typically used in a
reaction is so small that the contribution of the 1mM EDTA in very
small volume is negligible compared to the typical Mg++ reaction
buffer molarity. The same is true for template DNA in a typical PCR
reaction, but perhaps not in some restriction digests. Some people
use TE 10:0.1 (10 mM Tris-HCl, 0.1 mM EDTA pH 8.0) as a compromise.



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  #3  
Old 11-01-2004, 08:52 PM
Jose de las Heras
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Default solutions to storage oligonucleotides


"fabio" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...

It's better to store them in a buffered solution.
I store oligos at 100uM in 10mM Tris (pH8) as main stock, and at 5 or 10uM
as working stock to use day-to-day, again Tris 10mM.
TE is fine, given the kind of dilutions you do in the actual reaction you
won't have much EDTA, but as you mention, it may be a concern... so I just
use Tris for buffer, and leave EDTA out.

Jose


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  #4  
Old 11-01-2004, 09:22 PM
D.K.
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Default solutions to storage oligonucleotides

On Mon, 01 Nov 2004 16:08:30 +0100, fabio <[Only registered users see links. ]> wrote:


I store everythng in 1 mM Tris, pH somewhere around 8.0
(Qiagen's "EB" buffer diluted 10X; that's what I elute minis with).
1 mM gves enough to make sure pH is never acidic, yet it
does not have enough buffering capacity to make any contribution
in any enzymatic reaction). Addition of EDTA is good in theory but
offers little in practice - as soon as the solution is clean enough
(minipreps, gel-purified products, oligos, etc) there is no Mg2+ to
begin with and it's not going to maggically appear there. The oldest
primer I used that was stored this way was 5 years old - worked
same as when just made.

DK

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  #5  
Old 11-01-2004, 09:39 PM
Dr. Hiranya S. Roychowdhury
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Default solutions to storage oligonucleotides

All nucleic acids, whether oligos or genomic DNA or plasmids, should be stored
buffered. Store your primers in TE. A mM of EDTA is not going to be of any
consequence in your reactions.

Quoting fabio <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---
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  #6  
Old 11-03-2004, 11:12 PM
fabio
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Default solutions to storage oligonucleotides

D.K. wrote:

Thanks to all, so Tris-HCl or TE pH 8 are better than only water, now I
'm persuaded, but before closed the thread I have one further doubt:
becouse my routine PCRs are usualy RT-PCR and someone is of the type
one-step, I used to prefer water RNAse free (becouse treated with DEPC
and auocaved two times -two to be sure of destroy all DEPC), but I know
that DEPC demage Tris and, in the other hand the DEPC change the pH of
water if I remeber well, and the change is added to the one of the resin
"milli-Q" in the direction of acidity.
Work well without DEPC, even if good, isn't a garanty to don't have
RNAase, but maybe there are not choice.
What is your opinion?

Thanks again to all for your help.
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  #7  
Old 11-04-2004, 05:56 AM
Tom Knight
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Default solutions to storage oligonucleotides

fabio <[Only registered users see links. ]> writes:

Typically Tris buffers for RNA work are made with DEPC treated water
and solid tris. An alternative is to buy Tris buffers which are RNAse
free. These are available at final concentrations and at 1M, which
can be diluted with DEPC treated or other high quality water.

Also, high quality DI water systems with recirculation and UV
treatment produce RNAse free water without treatment. You have to be
careful that your solid Tris is not contaminated with sloppy
technique. You can also buy HPLC/reagent grade nuclease free water
and make solutions with that.
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  #8  
Old 11-07-2004, 02:15 PM
fabio
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Default solutions to storage oligonucleotides

Tom Knight wrote:

thanks!
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