[Zbrafish] Re: Help Help with microinjecting RNA

Bruce from WHOI via zbrafish%40net.bio.net (by bwoodin from whoi.edu)
Tue Oct 23 13:05:05 EST 2007


Tony-
I spoke with Matt Jenny, a post Doc in our lab, who has been using the
techniques you are attempting, and he says:
4 nL is OK but I would not go any higher....2 nL is better. The
location of microinjection is critical....need to be in the cell, or
if in the yolk very close to the cell. Do not use 0.1M KCl. I
microinject 2 nL with the mRNA in RNase free water with 0.001% phenol
red. The KCl may be throwing off the osmotic homeostasis.....my guess
is the eggs/yolk is swelling because of the influx of water. Also,
with that amount of eGFP you should be able to detect translation as
early as 3-4 hours post fertilization and the 24hpf embryo should
light up like a christmas tree.
Cheers,
Bruce



On Oct 22, 12:20 am, "cheuk.ch... from gmail.com" <cheuk.ch... from gmail.com>
wrote:
> Hi all,
>
> I am new to microinjection of zebrafish embryos and I need help.
>
> Briefly, I have injected 200pg of capped EGFP-coding RNA and the
> embryos show no expression. In addition, their yolk kind of leaks out/
> burst between 4hpf and 10hpf, thus killing the embryo.While uninjected
> ones look fine.
>
> To help with understanding the problem, I have detailed my protocol
> here.
>
> Capped RNA Synthesis
>
> I linearized pSP64TNE-EGFP at the Sal I site, Phenyl-Chloroform
> extracted the linearized template and then transcribe capped RNA with
> SP6 promoter using the Ambion mMessege Kit. The yield comes in at an
> acceptable 21ug with A260/280 at 1.94 and a A260/230 at 2.36. Running
> 200ng of the capped RNA in a 1% agarose gel gives me a sharp single
> band.
>
> Microinjection
>
> I diluted the capped RNA to 50ng/ul in 0.1M KCl and 0.05% PhenolRed.
> 4nl(200pg) was injected into each embryo, the embryos are recovered
> into 30% Danieau's Solution, and keep at 28.5C incubator.
>
> Any idea what can I be doing wrong here?
>
> Thanks a million for reading.
>
> Tony



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