1290 infinity 2 autosampler and binary pump

Cells were resuspended in TE buffer containing 62.5 μg/mL proteinase K (Invitrogen, AM2546), 62.5 μg/mL RNase A (Thermo Scientific, EN0531), and 0.5% SDS and incubated overnight at 37°C. Genomic DNA was purified by phenol/chloroform extraction and resuspended in RNase/DNase free water. 5 μg DNA was digested with 5 μL RNase H (NEB, M0297), 3 μL Hind III (Fisher, FD0504), 3 μL EcoRI (Fisher, FD0274), and 3 μL Bam HI (Fisher, FD0054) in RNase H buffer (NEB, M0297) overnight at 37°C. Digested DNA was purified using the GeneJET PCR Purification Kit (Thermo Scientific, K0702). DNA was further digested into single nucleosides using DNA Degradase Plus (Zymo Research, E2021). To quantitate DNA constituent base composition, a fit-for-purpose LC-MS/MS assay was implemented on a 1290 Infinity II Autosampler and Binary Pump (Agilent) and a SCIEX 6500+ triple quadrupole mass spectrometer (SCIEX). Chromatographic separation was conducted on an Inertsil ODS-3 (3 μm × 100 mm 2.1 mm) reverse phase column (GL Sciences) at ambient temperature with a gradient mobile phase of methanol and water with 0.1% formic acid. MRM transitions of all analytes and isotopic internal standards were monitored to construct calibration curves. We were able to quantitate 1 rN per 20,000 bases from 1 mg DNA.