[Zbrafish] Help Help with microinjecting RNA

cheuk.chung from gmail.com via zbrafish%40net.bio.net (by cheuk.chung from gmail.com)
Sun Oct 21 23:20:53 EST 2007


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



More information about the Zbrafish mailing list