Nature research paper: Mega-scale experimental analysis of protein folding stability in biology and design
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We prepared the photocrosslinking reaction solution using 100 mM NaCl, 20 mM Tris-HCl , 1 μM cnvK linker , 1 μM mRNA. The solution was incubated at 95 °C for 5 min, then slowly cooled down to 45 °C using a thermal cycler. Then the solution including the duplex was irradiated with UV light at 365 nm using a 6 W Handheld lamp for 15 min. At 40 μl scale , this produces crosslinked mRNA sufficient for 48 proteolysis reactions.We used the PUREfrex 2.
The cDNA amount for each specific sequence in the eluents was quantified by qPCR using SsoAdvanced Universal SYBR Green Supermix and specific primers for each sequence. The qPCR was performed using CFX96 Touch Real-Time PCR Detection System , and the qPCR cycles were determined by the CFX Maestro Software . Extended Data Fig.
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