To isolate genomic DNA (gDNA), 1×105 of both MC3T3-E1 and KRAB-MC3T3-E1 cells were resuspended in 50 μL of QuickExtract Solution (Lucigen - Epicenter, Middleton, WI, USA). The PCR reaction mixture was prepared with 10 μL of KAPA HiFi HotStart ReadyMix (2×) (Kapa Biosystems - Roche Applied Science Penzberg, DE-BY, DE), 0.5 μL of forward and reverse primers (10 μM) (F-ACTGATAAGGCTGACTTGCGGT R-CGAAGTTCCAGGGAGTGATGGT), 100 ng of gDNA and H2O up to 20 μl. The temperature cycling condition included an initial denaturation at 94 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 1 min and extension at 72 °C for 1min and a final extension for 10 min at 72 °C. The PCR products were mixed with 4 μL of Orange DNA Loading Dye (6×) (Thermo Fischer Scientific) and separated by 1% gel electrophoresis. The bands were recorded in the GelDoc UV (Bio-Rad Laboratories, Hercules, CA, USA) gel documentation system.
Orange dna loading dye 6
Manufactured by Thermo Fisher Scientific
Sourced in United States
Orange DNA Loading Dye (6×) is a pre-mixed solution used to prepare DNA samples for electrophoresis. It contains a tracking dye and glycerol to increase the density of the sample, allowing it to sink into the gel. The dye provides a visual indicator of the DNA migration during the electrophoresis process.
Automatically generated - may contain errors
2 protocols using orange dna loading dye 6
1
Genomic DNA Isolation and PCR Analysis
2
Analyzing mRNA Binding and Stability in Nanoparticles
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The determination of mRNA binding and its stability within the nanoparticles were analyzed by a gel retardation assay using 0.75% (w/v) agarose gel electrophoresis and tested for all mRNA complexed NPs with varying weight ratios (1:10, 1:20 and 1:30). To further induce a release of complexed mRNA, 5 μL heparin (30 mg/mL; Sigma-Aldrich, Darmstadt, Germany) was added to the mRNA complexed NPs and incubated for 15 min at room temperature. All samples were then mixed with 2 μL of orange DNA loading dye (6×, Thermo Fisher Scientific, Waltham, MA, USA), loaded into the agarose (Serva, Heidelberg, Germany) gel containing 3 μL of ethidium bromide (10 mg/mL; Sigma-Aldrich, Darmstadt, Germany) and run for 25 min at 90 V using 0.5× TBE buffer. The mRNA bands were then visualized with a UV illuminator (Fusion FX7 imaging system from Peqlab, Erlangen, Germany).
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