I'm using the TOPO TA cloning kit with TOP10 cells to clone a PCR product, which is amplified from RNA following reverse transcription. This is the first time I've done cloning, and I'm a bit confused. I want to check the orientation of my insert using restriction enzyme digestion, but not sure which way around it should actually be. Later I want to transcribe it using the T7 promotor site, which is downstream of the insert, so to get a transcript of the correct orientation does the insert need to be the wrong way around?! Or the same way around as it in the the original ssRNA?!
Submit your paper to J Biol Methods today!
TOPO cloning - direction of insert?
No replies to this topic