[Fwd: RE: [Zbrafish] About morpholino heating before injection]

Jon D.Moulton via zbrafish%40net.bio.net (by jmoulton from gene-tools.com)
Wed Jan 14 15:19:31 EST 2009


Hi all,

This is Jon from Gene Tools.  Here's our argument for water solution: 
(1) you can freeze-dry the oligo from a water solution, (2) you can 
prepare a sample for MALDI-TOF mass spectrometry from a water solution 
without ending up with a bunch of confounding salt-associated peaks in 
the spectrum, and (3) long ago at Antivirals Inc. (now AVI BioPharma 
Inc.) where Morpholinos were developed, Jim Summerton found that 
Morpholinos are more soluble in water than in buffers.

Some Morpholino sequences are not soluble at concentrations much above 1 
mM, which is why we recommend keeping Morpholinos in1mM stock 
solutions.  If you made the solution in water and for some reason needed 
a higher concentration, you could lyophilize the oligo and redissolve it 
at higher concentration.  Keeping a stock at a higher concentration 
encourages slow deposition of the oligo on the container walls, a 
process discussed below.

When Morpholino activity drops over time, we know where it goes.  The 
oligos associate with the walls of the container.  There is a 
destructive test for this: pipet the fluid form the container and pipet 
in 0.l N HCl, shake, wait and test by UV spectrometry.  Your missing 265 
nm absorbance will reappear because the acid protonates the oligos on 
the wall and they dissolve back into solution.  

Morpholinos are very chemically and biochemically stable, so room 
temperature storage is an option to help keep the oligos in solution -- 
I suggest keeping them in a dark box with the vial closure wrapped in 
Parafilm to discourage microbial contamination.  Never ice Morpholinos 
on the bench.  If you have some that have decreased solution 
concentration, you can try autoclaving them (a few times -- I hesitate 
to recommend routine autoclaving, as we haven't tested for stability 
through many rounds of heat sterilization).

Here is a link to the procedure for determining concentration by UV 
spectrometry.
http://www.gene-tools.com/files/determining_concentration.pdf

Let me know how I can help.

Regards,

 - Jon

    Jon D. Moulton, Ph.D.
    GENE TOOLS, LLC
    jmoulton from gene-tools.com
    www.gene-tools.com
    (541) 929-7840 x1201
    http://network.nature.com/group/morpholinos


-----------------------------------------------------------------

Hi,

We have used both Danieu's and water and haven't seen a difference in efficacy.  We currently use water.

In general, we make a very concentrated stock (50ug/ul) in water, aliquot into 5ul aliquots and store at -80C.  When we thaw one to make dilutions for injections, we keep the stock and dilutions at 4 degrees from that point on.  This is a relatively new procedure.  We used to store everything at -80 and thaw when needed, but we found our MOs stopped working.  Apparently others have seen this phenomena too, so Genetools recommended not freeze-thawing.  Hopefully Paul will chime in here.  I think it has something to do with the freeze thawing causing the MO to come out of solution?

We make dilutions for injection into the standard KCl/Phenol red injection buffer.  We do heat our dilutions to to 65 degree before injection.  I don't think we typically heat the stock before making dilutions, but it can't hurt.

I'd love to hear what other labs are doing.

Becky

---------------------------------------------------
Rebecca D. Burdine, Ph.D.
Assistant Professor
Dept. of Molecular Biology
Princeton University
Washington Road Mof 433
Princeton, NJ 08544 
 
Phone: (609) 258-7515
Fax: (609) 258-1343
Email: rburdine from princeton.edu
Admin Assistant: Cathy Falk (609) 258-1604

> -----Original Message-----
> From: zbrafish-bounces from oat.bio.indiana.edu [mailto:zbrafish-
> bounces from oat.bio.indiana.edu] On Behalf Of martam
> Sent: Wednesday, January 14, 2009 12:44 PM
> To: bionet-organisms-zebrafish from moderators.isc.org
> Subject: [Zbrafish] About morpholino heating before injection
> 
> Every people I know use a different procedure to dissolve morpholinos
> before inject them. So...
> Which is the correct procedure to dilute morpholino and prepare the
> injection mixture?
> I dissolve morpholino in Danieu buffer 1mM and freeze it.
> First question: which is the best choice to store stock solution of
> MO, -20°C or -80°C??
> Then, do I have to thaw the oligo before heating it to 65C for 10
> minutes?
> Once cooled to room, dissolved and diluted to the final needed
> concentration, is it better to heat the injection mix again?
> Thanks a lot.
> 
> 
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