I am interested in incorporating 120-150 A (Poly A tail) to increase the stability at the end of my vector to do invitro mRNA transcription. I don`t want to order long T primers. Does anyone knows any vector or other way to incorporate more Poly A at the end. Also what other factors can increase the mRNA stability.
Use a template-independent RNA polymerase. NEB used to sell poly(A) polymerase; look around their website.
Oh! I see. Polyadenylation is a post-trasncriptional event; there's no need to encode a polyA sequence in your vector. Rather, what you need is a polyadenylation signal. Grab one from your favorite eukaryotic expression vector and insert it into your construct.
Another, more involved alternative is available if you have two unique restriction sites after the CDS but before the site of transcriptional termination -if you are still bent on hard-coding a polyA without long dT primers. Cut the vector with the restriction enzyme whose site is closest to the CDS, then use a template-independent DNA polymerase (such as terminal transferase) to tail the ends with dATP, then cut with the other R.E., fill-in the polyA tail with Taq+dNTP and ligate.
hey matthew,Yes, the experiment is for invitro translation and then transfect into the cell. My goal is to transfect the mRNA into the cell and it`s half life should be more and translation efficiency should be as much as possible. So, i am considering to use sequences that can do the same .
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