Pcr strip tubes

Fresh Cas12 reactions were assembled by mixing reaction buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3), 45 nM AsCas12a V3 (IDT), 45 nM crRNA (IDT), 1 µM ssDNA Fluorophore-Quencher (FQ) Probe (IDT), and lyoprotectant. Prior to addition of FQ Probe and lyoprotectant, the mixture was equilibrated at 37 °C for 15 minutes. Upon addition of the analyte, the reaction was incubated at 37 °C and fluorescence was measured (490 nm Excitation, 525 nm Emission) using a Synergy H1 microplate reader (BioTek, USA) and Gen5 v. 2.09 (BioTek) software.
Lyophilized Cas12 reactions were assembled as described above and then flash frozen with liquid nitrogen in PCR strip tubes (Thermo Scientific, AB2000) with a small hole melted in the cap of each tube. The reactions were lyophilized using a VirTis BenchTop Pro lyophilizer (SP Scientific) at ≤100 mtorr and -80 °C overnight or until fully freeze-dried. Following lyophilization, tubes were packaged in Food Saver® bags with a desiccant card, vacuum sealed and stored at the indicated temperature. When ready for use, tubes were removed from the Food Saver® bags and rehydrated with the same volume of analyte as the mixture prior to lyophilization. Reactions were incubated at 37 °C and fluorescence was measured as described above.