Extended Data Fig. 9. ROSALIND with TMSD can be freeze-dried.
Unregulated reactions were lyophilized overnight with the addition of 50 mM sucrose and 250 mM D-mannitol as the lyoprotectants unless otherwise indicated. The lyophilized reactions were then vacuum-packaged in a light protective bag with a dri-card and kept in a cool, shaded area until usage (see Materials and Methods for the detailed protocol). Kinetic traces of rehydrated reactions after a, 1 day, b, 4 days and c, 7 days of storage are shown. There is a decrease in overall signal as well as in the response speed over time. To investigate the cause of the signal loss over time, the DNA signal gate alone was lyophilized overnight with or without the lyoprotectants, packaged and stored as described above. The DNA signal gate was rehydrated with the rest of the IVT components after d, 1 day, e, 4 days and f, 7 days. The response speed as well as the magnitude of the signal are maintained, indicating that the signal loss is likely due to instability of certain IVT components. To test this hypothesis, unregulated reactions with Tris-buffered NTPs, instead of NaOH-buffered NTPs, were lyophilized with the lyoprotectants, packaged and stored as described above. Kinetic traces of rehydrated reactions after g, 1 day, h, 4 days and i, 7 days of storage are shown. The signal loss is somewhat mitigated with Tris-buffered NTPs, but a similar degree of signal loss is observed for a long-term storage of lyophilized reactions. All data shown are n = 3 independent biological replicates each plotted as a line with raw fluorescence values standardized to MEF (μM fluorescein). Shadings indicate the average of the replicates ± standard deviation.