I have created capped, polyadenylated RNA in order to microinject it into zebrafish embryos. I would now like to make sure that what I've got is the right construct. How do I quantify the concentration of this RNA and how do I check the protocol has been correctly carried out? Thank you.
Hi Monika, whether you made it by in vitro system nanodrop is enough (normally, ug of RNA is made by this system). Although, if you added polyA with polyA polymerase from ambion you should see you polyA RNA as a smear on FA-agarose gels in compare to only capped RNA due to different amounts of As added by it. Otherwise, you should see a sharp band in same gels if you plasmid contains already polyT at 3end (defined amounts of As). Good luck!.
It was discontinued 2 days ago, but the ExactStart kit from EpiCentre would be useful for you. It will help you do a 5' and 3' RACE to prepare a library of only capped and poly(A) RNA. Unfortunately, the crucial decapping enzyme TAP doesn't exist anymore. There might be a kit or two still somewhere in the supply chain.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA