Extended Data Fig. 2: Gel electrophoretic analysis and flow cytometric validation of mPlum_O6-MeG and BFP_U reporter plasmids. | Nature Protocols

Extended Data Fig. 2: Gel electrophoretic analysis and flow cytometric validation of mPlum_O6-MeG and BFP_U reporter plasmids.

From: Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

Extended Data Fig. 2

a, Analytical digestion and flow cytometric validation of mPlum_O6-MeG plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_mPlum_C207G/T208C ccDNA; lane 3: pMax_mPlum_C207G/T208C after a 45-min PspOMI digestion at 37 °C, resulting in linear pMax_mPlum_C207G/T208C starting plasmid; lane 4: mPlum_O6-MeG plasmid after T5 Exo and PEG purification steps; lane 5: mPlum_O6-MeG after a 45-min PspOMI digestion at 37 °C, in which the O6 group on the guanine blocks linearization by PspOMI, leaving predominantly ccDNA product (note: upon extended digestion or when excess enzyme is present, some linearized DNA will result). At right: flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in MGMT-deficient TK6 cells compared to TK6 cells complimented with stable MGMT expression. b, Analytical digestion and flow cytometric validation of BFP_U plasmid. Lane 1: NEB 1-kb MW ladder; lane 2: pMax_BFP_A191G ccDNA; lane 3: pMax_BFP_A191G ocDNA; lane 4: pMax_BFP_A191G after a 5-min UDG digestion at 37 °C, followed by a 30-min APE1 digestion at 37 °C; lane 5: BFP_U after a 5-min UDG digestion at 37 °C, followed by a 30-min APE1 digestion at 37 °C, resulting in UDG excising the incorporated uracil, followed by APE1 nicking the abasic site, resulting in an upward gel mobility shift; lane 6: BFP_U plasmid after T5 Exo and PEG purification steps. At right: Flow cytometric quantitation of percent reporter expression and normalized relative reporter expression in WT MEF cells compared to UNG−/− MEF cells. Error bars represent s.e.m. from three to four biological replicates; differences of statistical significance (*, P < 0.05; **, P < 0.005; ***, P < 0.005) were determined by unpaired two-tailed t test.

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