Hello,
I have experience producing transgenic zebrafish with CRISPR and the CAS9 mRNA would be polyadenilated with a Poly(A) tailing kit after in vitro transcription. However, with Tol2, the papers I have read don't mention a polyadenilation step for the transposase mRNA. I am using plasmid pT3TS-Tol2 to generate mRNA but I also have pCS2FA-transposase. Is it that transposase mRNA doesn't need polyadenilation?
Hi Carlos,
I have always added a CAP and the PolyA tail to my transposase and it has worked nicely. I have used an UBI promoter as a positive control, which has shown me that my transposase is working. I have used NEB reagents and protocols:
https://www.neb.com/products/m0276-ecoli-poly-a-polymerase
Hope that helps!
P.S. I'm trying to do CRISPR in zebrafish and I'm collecting protocols, any chance of obtaining yours? :)
As the person "responsible" for making pT3TS-Tol2, I'd say yes. I very strongly recommend using Ambion's mMessage mMachine T3 kit. A single transcription reaction will give you enough Tol2 mRNA for years to come, since you only need very little for each injection batch.
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