- The DNase I assay was adapted from the standard protocol of the - Picogreen Assay kit (ThermoFisher Scientific, 2022). This kit uses a - reaction buffer containing 1 M Tris-HCl (pH 7.5), 1 M MgCl2, and 1 M - CaCl2 (ThermoFisher Scientific, 2022), which will be referred to as - the manufacturer’s buffer. 0.05 U/mL and 0.1 U/mL DNase I solutions - were prepared in the manufacturer’s buffer, the TE buffer and the - HEPES buffer separately for use in the activity assay. Buffer DNase - concentration is given in units of DNase activity per mL (U/mL), - with the activity unit defined as the complete degradation of 1 µg - of plasmid DNA at 37°C by one unit of DNase I in 10 minutes - (ThermoFisher Scientific, 2011). The HEPES buffer was also used for - thiolation treatment and conjugation to the liposome. + The DNase I assay was adapted from the standard protocol of the Picogreen Assay kit + (ThermoFisher Scientific, 2022). This kit uses a reaction buffer containing 1 M + Tris-HCl (pH 7.5), 1 M MgCl2, and 1 M CaCl2 (ThermoFisher + Scientific, 2022), which will be referred to as the manufacturer’s buffer. 0.05 U/mL + and 0.1 U/mL DNase I solutions were prepared in the manufacturer’s buffer, the TE + buffer and the HEPES buffer separately for use in the activity assay. Buffer DNase + concentration is given in units of DNase activity per mL (U/mL), with the activity + unit defined as the complete degradation of 1 µg of plasmid DNA at 37°C by one unit of + DNase I in 10 minutes (ThermoFisher Scientific, 2011). The HEPES buffer was also used + for thiolation treatment and conjugation to the liposome.
- An aqueous working solution of the Quant-iT™PicoGreen™ dsDNA Reagent was - prepared in the TE buffer by using the buffer to dilute it 200 fold. A 2 µg/mL - stock solution of lambda dsDNA was prepared in the TE buffer as well by using - the buffer to dilute it 50 fold. In a 96 well plate, the controls were created - by adding water, the manufacturer's buffer, the TE buffer, and the HEPES buffer - to twelve wells such that each solution type had three wells of 80 µL each. For - the other treatments, 40 µL of the aqueous working solution of the Quant-iT™ - PicoGreen™ dsDNA Reagent was added each well, followed by the designated amount - of buffer for each treatment shown in Table 1 as well as 4 µL of the stock - solution of lambda dsDNA. The wells were incubated for five minutes at room - temperature with protection from light before adding the corresponding volume of - DNase I indicated in Table 1. Fluorescence was measured at an excitation - wavelength of about 480 nm and an emission wavelength of about 520 nm to - determine enzyme activity using a fluorometer-spectrophotometer in kinetic mode - every 2 minutes for at least 10 minutes. These treatments were performed three - times to obtain a triplicate. + An aqueous working solution of the Quant-iT™PicoGreen™ dsDNA Reagent was prepared in + the TE buffer by using the buffer to dilute it 200 fold. A 2 µg/mL stock solution of + lambda dsDNA was prepared in the TE buffer as well by using the buffer to dilute it 50 + fold. In a 96 well plate, the controls were created by adding water, the + manufacturer's buffer, the TE buffer, and the HEPES buffer to twelve wells such that + each solution type had three wells of 80 µL each. For the other treatments, 40 µL of + the aqueous working solution of the Quant-iT™ PicoGreen™ dsDNA Reagent was added + each well, followed by the designated amount of buffer for each treatment shown in + Table 1 as well as 4 µL of the stock solution of lambda dsDNA. The wells were + incubated for five minutes at room temperature with protection from light before + adding the corresponding volume of DNase I indicated in Table 1. Fluorescence was + measured at an excitation wavelength of about 480 nm and an emission wavelength of + about 520 nm to determine enzyme activity using a fluorometer-spectrophotometer in + kinetic mode every 2 minutes for at least 10 minutes. These treatments were performed + three times to obtain a triplicate.
- Table 1: Designated buffer and DNA volumes for each treatment. + Table 1: Designated buffer and DNA volumes for each treatment.
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