In order to directly compare the DNA yield per mm3 of dissected tissue, identical areas were dissected by digitally guided microdissection or manual macrodissection on adjacent sections. Crude lysate preparation was performed as described above, and samples were stored at 4°C. DNA yield assessment was performed simultaneously for all samples. DNA concentrations of unpurified crude lysate samples were determined using PicoGreen fluorescence according to the manufacturer's instructions (Molecular Probes®, Thermo Fisher Scientific, Waltham, MA, USA). DNA quality was also determined by fluorescence resonance energy transfer (FRET) real time PCR amplification of a 200 base pair sequence from the beta globin gene (HBB) (forward primer 5’ - ACA CAA CTG TGT TCA CTA GC – 3’ and reverse primer 5’ - CAA CTT CAT CCA CGT TCA CC – 3’ with a fluorescein labeled probe: 5′ - GGA GAA GTC TGC CGT TAC TGC C/dye - 3′ and LC Red 640 probe: 5′- dye/AGA CTT CTC CTC AGG AGT CAG GTG CAC CAT G - 3′) (LightCycler®, Roche Diagnostics, Indianapolis, IN, USA).
Picogreen fluorescence
Manufactured by Thermo Fisher Scientific
Sourced in United States
PicoGreen is a fluorescent dye used for quantifying double-stranded DNA (dsDNA) in solution. It binds to dsDNA and exhibits a strong fluorescent signal upon excitation, allowing for the sensitive detection and measurement of DNA concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Genelute mammalian genomic dna miniprep kit, by Merck Group (2 mentions)
Lightcycler, by Roche (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Flurotrac 200 black plate, by Greiner (1 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Mab3034, by Merck Group (1 mentions)
6 protocols using picogreen fluorescence
1
Comparative DNA Yield Analysis
2
Automated ALE Experiments with JCVI-syn3A
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ALE experiments were conducted with automated devices9 (link) that serially propagated batch cultures of JCVI-syn3A between growth tubes with 15 mL of SP4-KO medium (containing 17% v/v KnockOut™ Serum Replacement [GIBCO] in lieu of fetal bovine serum in SP4), well aerated through mixing and kept at 37°C. Ten replicate lineages were started from the JCVI-syn3A ancestor and for each lineage 150 ul of culture was passed to a fresh tube once daily (∼6.6 generations/day), switching to 30 ul passage volume (9 generations/day) after 200 generations and continuing for a total of 400 generations.
Strains were isolated for characterization by plating of evolved populations and random selection of individual clones. Clones were inoculated into SP4-KO medium at 37°C and sample timepoints were taken regularly during the cultures’ period of exponential growth. Samples were centrifuged, cells were lysed, and DNA content was quantified via PicoGreen fluorescence (Thermo Fisher) as described previously.2 (link) The slope of log(DNA fluorescence) vs. time was then calculated to determine culture growth rates; all growth curve data had a line of best fit with R2>0.99.
Strains were isolated for characterization by plating of evolved populations and random selection of individual clones. Clones were inoculated into SP4-KO medium at 37°C and sample timepoints were taken regularly during the cultures’ period of exponential growth. Samples were centrifuged, cells were lysed, and DNA content was quantified via PicoGreen fluorescence (Thermo Fisher) as described previously.2 (link) The slope of log(DNA fluorescence) vs. time was then calculated to determine culture growth rates; all growth curve data had a line of best fit with R2>0.99.
3
Fluorometric Analysis of Alkaline Exonuclease
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Alkaline exonuclease (UL12) activity was monitored by the PicoGreen assay as described previously (16 (link), 63 (link)). Briefly, 25-μL reaction mixtures containing 20 mM Tris-HCl (pH 8.2), 40 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol (DTT), 10 nM UL12, and 1.6% DMSO or 20 μM inhibitor in 100% DMSO were preincubated at 37°C for 10 min prior to addition of DNA substrate. The nuclease reaction was initiated by adding 1 nM linearized pUC19 DNA, the mixture was incubated at 37°C for an additional 5 min, and the reaction was quenched by the addition of 10 μL 2.5 mM EDTA (pH 8.0). The quenched reactions were processed for PicoGreen fluorescence according to the manufacturer’s protocol (Invitrogen). Briefly, each quenched reaction was diluted to 100 μL by the addition of Tris-EDTA (TE [pH 7.5]), mixed with 100 μL of PicoGreen (1:200 diluted in TE buffer), and transferred to a 96-well Flurotrac 200 black plate (Greiner Bio-One). Fluorescence was measured using a SpectraMax 3 plate reader with excitation and emission wavelengths of 480 and 520 nm, respectively.
4
Genomic DNA Purification and Quantification
5
Quantitative PCR Analysis of DNA Damage
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The quantitative PCR (QPCR) reaction was performed as described previously.12 Briefly, after genomic DNA extraction and quantification by PicoGreen fluorescence (Invitrogen, Carlsbad, CA, USA), the DNA was used as template for the PCR reaction. For these, TaKaRa LA PCR (Takara Bio Inc., Mountain View, CA, USA) and the sequencing primers 5′-TGGAAACCCTGTGGGCGGATAATA-3′ and 5′-CTCCAGGCCTAAGGAGCAGCAGAA-3′ were used. The PCR products were quantified by PicoGreen fluorescence, and the cisplatin-treated (50 μM) or BSO-treated samples (100 mM) were divided by control sample. The resulting ratio was the relative amplification of damaged to control samples, and the DNA lesion frequencies were estimated based on the Poisson distribution.
6
Quantifying DNA Replication and Damage
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