I'm trying to do RNA pull down assay with Biotin RNA Labeling Mix (Roche) . The Kit require the sequence under T7 promoter. Now I only have the cDNA without T7 promoter. If I use PCR to add the promoter, how do I make sure the primer amplify the right sequence? Besides, is there any recommend for a plasmid with T7 promoter?
The pCR4-TOPO vectors have the T7 promoters. I imagine a simple method would just be to insert your cDNA into one of them. Once your cDNA is in the plasmid, you can perform PCR using M13 primers to amplify a linear fragment. This linear fragment will have the T7 promoter and could be used for RNA transcription.
The problem with this method is you cannot control the orientation of your cDNA insert, so you would need to sequence several colonies and find one where the T7 promoter is properly aligned with your sense or antisense sequence.
I hope that helps
https://www.thermofisher.com/no/en/home/life-science/cloning/topo/topo-ta-cloning/topo-ta-for-sequencing.html
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