Second strand synthesis yields.

<p>(A) Box plots of second strand syntheses yields by starting amount of ssDNA indicate scalable and reliable yields. (B) Box plots comparing yields of lesion-free versus lesion-containing (8-oxoG, 5-OHU, or DHU) constructs, per 40 μg of ssDNA, or as expected per 40 μg of ssDNA if actual starting ssDNA amount was less (20–30 μg). Similar yields are obtained for lesion-free and lesion-containing constructs. Whiskers extend 1.5 times the interquartile range from the 25^th and 75^th percentiles, which are indicated by box limits. Center lines indicate the medians and circles represent the values for individual preparations. Determined by R software.</p

Phenotypic consequences of 5-hydroxyuracil in Neil1^-/-Neil2^-/- MEFs.

<p>(A) Schematic of the construct, designed such that mutagenic bypass by RNA polymerase of 5-OHU produces the G12D mutant of K-Ras, leading to the activation of downstream oncogenic signaling. (B) Representative Western blot showing sustained increase in AKT phosphorylation at 24 hours post-nucleofection, with each sample loaded twice serving as a technical replicate. (C) Western blot quantification of two biological replicates, each containing two technical replicates, of pAKT and pERK relative to G12D positive control. The increase in AKT phosphorylation is statistically significant, as determined by a t-test. Error bars represent the standard error of the mean.</p

Lesion-containing construct quality controls.

<p>(A) Schematic of the Fpg nicking assay. Fpg cleaves damages, such as 8-oxoG and 5-OHU, leaving a single-strand break, converting the construct from covalently closed (cc) to nicked form. (B) Lesion structures. (C) Representative images of Fpg and Nth nicked T5 exonuclease-treated lesion-containing and lesion-free control constructs. Fpg cleaves 8-oxoG, 5-OHU, and DHU, nicking the lesion-containing constructs almost entirely, but not the lesion-free controls, and Nth cleaves DHU.</p

Culture density at time of helper phage infection predicts ssDNA yield.

<p>Scatter plot of large-scale (200 mL) ssDNA preparations infected at various MOI greater than one, following anion-exchange column purification. Black line represents the local polynomial regression (loess) curve and grey area the 95% confidence interval, as determined by R software.</p

Optimization of the DH12S <i>E</i>. <i>coli</i> culture and phage infection conditions for phagemid ssDNA yield.

<p>(A) Schematic of the experimental design for the systematic analysis of the effect of culture OD₆₀₀ at phage infection and multiplicity of infection (MOI) on ssDNA yield. Cultures were infected at varying OD₆₀₀ or MOI and ssDNA yields were determined by proteinase K digestion of precipitated phage particles, followed by agarose gel electrophoresis. (B) OD₆₀₀ at phage infection predicts ssDNA yield. (C) Increasing MOI beyond that which is necessary to infect all cells does not improve ssDNA yield. (D) Gel quantification of two biological replicates, each containing three technical replicates, of cultures infected at varying OD₆₀₀ and identical MOI. (E) Quantification of ssDNA yields from cultures infected at varying MOI. Averages include two biological replicates, each containing three technical replicates. Error bars indicate the standard deviation. (F) Cultures that otherwise produce high yields of phagemid ssDNA, infected at MOI > 2.5 or 10, do not yield ssDNA when diluted 2 hours following phage-infection.</p

Optimization for DNA integrity and mammalian transfection.

<p>(A) Schematic representing T5 exonuclease digestion of nicked, linear, and ssDNA. (B) Representative gel electrophoresis of a construct with and without T5 exonuclease treatment prior to purification and after purification. (C) Construct yields after T5 exonuclease treatment after initial purification (after) or directly in the second strand synthesis reaction (before), relative to non-T5 exonuclease treated construct (none). Error bars represent the standard deviation. (D) Live cell images of Ogg1^-/- MEFs nucleofected with EGFP construct treated or not treated with T5 exonuclease or EGFP bacterial plasmid maxiprep, and stained with Hoechst 33342 dye. T5 exonuclease digestion of nicked and linear construct does not improve transfection efficiencies.</p