3 replies to this topic

[moerae]

Hi,

I'm trying to over-express a protein in zebrafish and I have them in a vector that doesn't have a polyA signal. I will eventually clone it into a vector that has a signal, but I want to just see whether the expression of the protein is correct first. I was going to do an RNA run off and inject into the zebrafish. Will the lack of polyA signal affect the expression of protein?

[bob1]

Possibly, but not all mRNAs have a poly A tail, so it may be OK.

[moerae]

Thanks for the reply. I'm going to do in vitro transcription using T7/T3 RNA polymerase and the kit I have only adds the 5' cap. Am I right to assume that I'll need a specific kit to add a polyA tail to the end of the RNA transcript?

[LabLackey]

Usually, yes.  I know we have bought a poly(a) polymerase from NEB that works quite well.  I think by adenylating your transcript you will see an increase in protein expression.  Let us know how it goes.