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TOPO cloning - direction of insert?

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#1 katcvet



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Posted 06 June 2011 - 07:13 AM

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?!

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