[Zbrafish] About morpholino heating before injection

[Burdine, Rebecca D]

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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