Hi again. I was looking at an ad for USB's Lysate RNAase Protection Assay.
This uses the same lysis procedure as in the AGPC RNA prep, except that there
is no 2-mercaptoethanol added. I have tried using AGPC lysates for solution
hybridizations and they do not work, maybe the mercaptoethanol is the reason.
Why is there mercaptoethanol at all? I think, without reducing agents, RNAse
is inhibited in GnSCN down to about 2 M, and with reducing agents, inhibition
is complete down to about 1 M. Is this the only reason for using 2-ME? Can I
get away with omitting it in my regular RNA preps if I keep the GnSCN conc.
above 2M? I'd like to try doing Poly(A)+ selection in the crude lysates if
I can get away with it.
Thanks for advice. Daniel Kim