Nanodrop spectrometer spectramax id5 multi mode microplate reader

The crRNAs extended with 11-mer DNA (No. 12 in Table 1) [27 (link)] were chemically synthesized by Integrated DNA Technologies. For obtaining crRNA to detect the stn gene, nuclease-free water (5.5 μL), 10 × reaction buffer (1.5 μL), NTPs (1.5 μL of each; final concentration: 7.5 mM), 3.6 μM IVT template DNA (5.5 μL; No. 14 in Table 1), and T7 RNA Polymerase Mix (1.5 μL) were mixed. The mixture was incubated at 37 °C for 16 h and then treated with DNase I to remove the remaining IVT template DNA; nuclease-free water (70 μL), 10 × DNase I buffer (10 mM Tris–HCl, 2.5 mM MgCl₂, 0.5 mM CaCl₂; pH 7.6, 25 °C) (10 μL), and 2 U/μL DNase I (2 μL) were added to 20 μL of the IVT product, and the mixture was incubated at 37 °C for 15 min. Finally, crRNA was purified using the Monarch RNA Cleanup Kit (New England Biolabs). The purity and concentration of the synthesized crRNA were evaluated using a Nanodrop Spectrometer (Spectramax iD5 multi-mode microplate reader; Molecular Devices, San Jose, CA, USA) [27 (link)].