At this stage of my project, I am quickly progressing towards being able to inject nfe2 knockout zebrafish embryos with mRNA coding for alas2.
Last week, I isolated DNA plasmids from E.coli that contained alas2 in their plasmid. I then linearized the plasmid using restriction enzymes, so that it could be used to create the capped mRNA necessary for microinjections.
When using another kit to create the capped mRNA, I’ve run into a small snag. The DNA digest produced linearized DNA with a concentration of 43.8 ng/ul, and the kit requires me to use approximately 0.1-1ug of DNA while only sticking to a total reaction volume of 20ul. There are a few things that we could do here. We could scale the reaction up (which is a method recommended by the kit if a situation like this arises), we could use a smaller concentration of DNA (because we would have to use 22ul of the linearized DNA to get the 1ug required by the kit), lastly we could perform the DNA digest again (where we use more of the DNA plasmid recovered from the E.coli (I.e. 5ug instead of the 1ug required by the procedure). In the end, we chose to use a smaller concentration of linearized DNA (6ul = 0.262ug of DNA) which would allow us to stay in the bounds of a 20ul reaction mixture.
When I’ve produced the necessary capped and polyadenylated mRNA, I’ll be able to collect embryos and inject them with it. In the last month or so that I’ve been breeding the nfe2 knockout fish, the number of embryos that they produce fails in comparison to other zebrafish (such as the AB zebrafish). Therefore, I’ll be continuing to practice my technique on microinjections to increase my efficiency of performing successful injections.
For the remainder of the week, I’ll be troubleshooting the mRNA kit if my first attempt doesn’t quite work out, microinjecting, and continuing work on writing my thesis introduction.
On a side note I’m really interested in the biology talk this week about genetically controlling pluripotency of embryonic stem cells!