How to inactivate Yeast Poly A polymerase

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How to inactivate Yeast Poly A polymerase

Postby semmelmk » Sep 16 2015 7:14 pm

I have biotinylated RNA and tail it with Yeast Poly A Polymerase (PAP). After tailing I want to bind to the RNA to magnetic Streptavidin beads and wash the RNA.
I need to find a way to completely inactivate the PAP without heat killing or chelating (downstream in my process I use different RNA oligos that must not get tailed). Actually I realized that heat killing (65C for 10 mins.) does not inactivate PAP completely.

Maybe I could deactivate the enzyme in the washing step after binding to the beads. I tried 1%SDS in wash buffer or 6 M Guanidine. Both did not do the job. Any idea or suggestions? The enzyme seems to be bound to the RNA or beads and therefore stabilized.

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