So right now I am working on using Sanger Sequencing to characterize some viral RNA isolates, and I am focused on creating amplicons for the 5' and 3' ends of my genome. I plan to use the Takara Biosciences SMARTer 5'/3' RACE kit, and for the 5' end I think I'll use random primers instead of the provided 5' CDS primer A. Problem is for 3' end, I'm not sure if I can use random primers, given that the kit's manual didn't say anything about using this method to create 3' cDNA. I want to add a 3' end, but given the size of my genome is 10,000 bp, I don't think that's possible. So my crazy idea is to create a primer that will anneal near the 3' end, and substitute it as the 3' CDS primer A. Does anyone have experience with this? Please let me know!
The SMARTer 5'/3' RACE Kit is a powerful tool for amplifying the ends of RNA transcripts, including viral RNA, especially when you're interested in studying the 3' ends that include the poly(A) tail. Here are the steps and considerations for using this kit effectively with viral RNA that has a poly(A) tail:
1. RNA Isolation
2. Poly(A) Tail Enrichment
3. Reverse Transcription
4. Amplification of cDNA
5. Optimization
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