Combine all your components in a microcentrifuge tube: Pipet 35 microliters Rabbit Reticulocyte Lysate System, 0.5 microliters of the amino acid mixtures, 1 microliter of RNasin, 2 microliters of mRNA and 1 microliter of tRNA together, and mix. The Rabbit Reticulocyte Lysate System includes all the molecules that are found inside our cells and required for translation, including ribosomes and initiation factors. This step will prepare everything ready for the translation. Your mRNA molecule attaches to a ribosome, and the initiation factors assemble. Molecules called tRNAs are associated with their amino acids and are ready to build the protein.
Heat the reaction mixture to 30 degrees Celsius. The translation will start during this step. The message in the mRNA is "read" by the tRNAs. Each tRNA carries an amino acid, and these amino acids are joined together to form the protein.
Wait 90 minutes for the translation to proceed. The tRNAs keep bringing new amino acids according to the mRNA message. Once the mRNA is fully read, the translation ends and the protein is ready.
Purify your protein by SDS-PAGE. It is ready for functional tests.