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.

[Jon Moulton]

Hello Martam,

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. ]

Heating to 65C for 10 minutes and cooling to room temperature before use helps dissolve any oligo which might have come out of solution -- it's a good routine. You can put frozen stocks into the 65C bath, they will rapidly melt. To allow a little extra time for melting, heating the stocks in the 65C bath for 11 or 12 minutes if they are frozen would be OK. You can probably skip reheating the solution after making dilutions for injection unless you are clogging needles. If your needles clog, try a final 65C warm-up to help dissolve any precipitate.

Let me know how I can help.

Regards,

- Jon

[Jon Moulton]

LOL - I see that my response to the Zebrafish newsgroup already made it here in a different thread. I sure don't want to quell discussion on this with my response. Please, if you microinject Morpholinos, chime in with your experience!

[Jon Moulton]

Almost four years later -- we have changed to recommending room temperature storage of Morpholino solutions. The freeze-thaw cycles tend to drive the oligo out of solution, so since they are chemically stable at room temperature it's best to avoid freezing. For longer-term storage, you can heat the solution, aliquot it and freeze-dry the aliquots (but do true lyophilization, don't just put them into a SpeedVap). Freeze-dried the oligos will be stable for many years.

[Jon Moulton]

Here's a page discussing dissolution and storage of Morpholinos:
[Only registered users see links. ]