Posted 03 December 2001 - 10:00 PM
I'm not sure if you can do single stranded RNA ligation, but I have another idea. What if you did first strand synthesis on the RNA. Then, do a second strand synthesis (protocol in maniatis or current protocols). Next, blunt the ends of the double stranded cDNA you produced (just to be sure). Next, make double stranded linker DNA that has a specific primer site in it and do a ligation with your double stranded DNA. Finally, do a PCR reaction with this ligation as a template and the primer you designed into the linker DNA, and a specific primer you choose from sequence you already know. You will probably get several bands, but hopefully you can estimate the size of the band you want. Gel purify that band and sequence it directly or subclone it. There are protocols in maniatis and current protocols on how to do each of these steps. In theory, I think this should work- any thoughts from anyone else?