Hi, I designed a guide RNA sequence that would be used with cpf1 (from NEB) for a knock-out experiment. Before transforming the RNP complex to my culture, there is an incubation step with guide RNA and Cpf1 which allow them to form a RNP complex. I am wondering if the melting temperature (Tm) of my guide RNA needs to be considered when performing this incubation step, since the protocol from NEB simply says incubate in room temperature.
Hi Justin, No answer for a long time. It is very unlikely, that you should be concerned about Tm ( as it for RNA-DNA interaction latter on). For your complex formation the interaction will be protein-DNA and as far as I remember it is protein interaction with phosphate backbone mainly, so it shouldn't be sequence specific, or very little sequence dependence. So Room T it is!
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