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

[Fwd: RE: [Zbrafish] About morpholino heating before injection] - Zebrafish Forum

[Fwd: RE: [Zbrafish] About morpholino heating before injection] - Zebrafish Danio Rerio Forum. Discuss zebrafish, aquarium culture and molecular biology lab specimens.


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Old 01-14-2009, 08:19 PM
Jon D.Moulton
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Default [Fwd: RE: [Zbrafish] About morpholino heating before injection]



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.
[Only registered users see links. ]

Let me know how I can help.

Regards,

- Jon

Jon D. Moulton, Ph.D.
GENE TOOLS, LLC
[Only registered users see links. ]
[Only registered users see links. ]
(541) 929-7840 x1201
[Only registered users see links. ]


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

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: [Only registered users see links. ]
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