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RNA ligation


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#1 anonymous

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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?

#2 Leong Wai Fook

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Posted 03 December 2001 - 10:00 PM

Is there such thing as RNA ligation? I have a RNA virus genome to sequence but being not circular, I have problem getting the ends of the genome. I was thinking if there is a possible way of circularizing the RNA genome then I will be able to do RT using random primers followed by outward PCR using the primers from the known region.

Thanks.


#3 Leong Wai Fook

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Posted 04 December 2001 - 10:00 PM

Thanks for your suggestion. Looks very feasible. Just to clarify something. Do I do 1st strand synthesis using random primer or oligodT primer ? BTW the genome size of the viral RNA is about 7kb, is it possible to stretch RT to that length?

#4 anonymous

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Posted 06 December 2001 - 10:00 PM

I think optimally, oligo dT could go that far, but I would stick with random primers. Whenever I am cloning things from rtPCR, I consistently have had more success with random primed cDNA compared to oligodT. Good luck!




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