[Zbrafish] Re: Morpholinos (Burdine, Rebecca D)

Lavinia Sheets via zbrafish%40net.bio.net (by sheetsl from ohsu.edu)
Fri Apr 23 15:49:18 EST 2010


Just writing to say ditto about the 65C degree incubation.  I've stored my MO stocks both frozen and at RT and have found that, regardless of how I store them, incubating at 65C for a few minutes prior to making a working solution is critical to maintaining morpholino efficacy.

Good Luck:)

Lavinia Sheets
Post Doctoral Associate
Teresa Nicolson Lab
HHMI/ Oregon Health & Science University 
________________________________________
From: zbrafish-bounces from oat.bio.indiana.edu [zbrafish-bounces from oat.bio.indiana.edu] On Behalf Of Wilson Clements [wclements from ucsd.edu]
Sent: Friday, April 23, 2010 10:28 AM
To: zbrafish from oat.bio.indiana.edu
Subject: [Zbrafish] Re: Morpholinos (Burdine, Rebecca D)

Dear Rebecca,

Morpholinos crashing out of solution sounds like a problem.  I agree
that it is important to resuspend at a high(er than recommended)
concentration.  Here is what I do (we have not had precipitation
problems):

When the morpholinos arrive, I resuspend them at 25mg/ml (~3mM) in
DEPC H2O.  (DEPC H2O permits later co-injection of mRNA, if
necessary).  25 mg/ml generally means resuspending in approximately
100ul.  I subaliquot this 100ul to 10 X 10ul, and store the aliquots
at -80C.  This concentration allows for achievable injection
concentrations of up to 20mg/ml, or 20ng assuming a 1nl injection drop.

I work with a single aliquot until it is exhausted, making up new
dilutions each time I inject.  Since most of my morpholinos work at
concentrations between 2 and 7 ng, this generally means somewhere
between 5 and 15 freeze/thaw cycles.

IMPORTANT:  I always heat the thawed aliquot 3 minutes at 65C, and
spin down before making a new dilution for injection.  In the past I
did not heat the morpholinos and I saw an apparent decrease in the
activity of the morpholino using the same dosage that was previously
effective.  My interpretation of the heating requirement is that the
morpholinos adopt an inactive conformation that is relieved by the
heating.

I do not use "Danieau" buffer. DEPC H2O works fine for me.

Best,
Wilson
--------------------------------------------------------------------------------------------
Wilson Clements, Ph.D.

wclements from ucsd.edu

Dept. of Biology
Section of Cell and Developmental Biology
University of California at San Diego
9500 Gilman Dr.
Natural Sciences Building 6105
La Jolla, CA 92093-0380

TEL    (858) 534-6955
LAB    (858) 822-4658
FAX    (858) 822-5740


On Apr 23, 2010, at 10:03 AM, zbrafish-request from oat.bio.indiana.edu
wrote:

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>   1. Morpholinos (Burdine, Rebecca D)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Fri, 23 Apr 2010 12:05:22 -0400
> From: "Burdine, Rebecca D" <rburdine from Princeton.EDU>
> Subject: [Zbrafish] Morpholinos
> To: <bionet-organisms-zebrafish from moderators.isc.org>
> Message-ID:
>       <790DAC09623D0A47ACDB126E3C52D8B6642B8D from MBCLUSTER.pu.win.princeton.edu
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>
> Hi everyone,
>
>
>
> We are having the issue that our newer MOs are falling out of
> solution,
> even at 4 degrees.  We are being advised to keep them at RT, but
> others
> have told me they evaporate too quickly at RT.  Our other issues are
> that we have historically always resuspended to make concentrated
> stocks
> (which is compounding our problem) and we transfer the stocks to
> epitubes.
>
>
>
> My questions are the following:
>
>
>
> 1.     Has anyone come up with a better medium for resuspending that
> is
> less prone to having the MOs fall out of solution?
>
>
>
> 2.     We are reluctant/worried about resuspedning at a lower
> concentration.  For example, the sqt MO is published as being injected
> at 7ng to get the phenotype and we have found this to be accurate.
> This
> is going to be very hard to reproduce with a 1mM stock.  Has anyone
> found that when they resuspended at a lower concentration that the MO
> actually worked better?
>
>
>
> 3.     Does anyone have any storage advice that has worked well for
> them
> and yet prevents massive evaporation?
>
>
>
>
>
> Thanks,
>
> 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) 258-6730
>
> Email: rburdine from princeton.edu <mailto:rburdine from princeton.edu>
>
> Admin Assistant: Anna Schmedel (609) 258-5028
>
>
>
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