High Pure PCR Template Preparation Kit

Protocol tips

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	Before starting the purification, warm the Elution Buffer to +70°C.

Protocol tips
Before starting the purification, warm the Elution Buffer to +70°C.

Publication protocol

DNA isolation
The culture media and placental pieces were collected at times zero, 6 and 21 hours. To assess cell-free DNA (cfDNA) released by the placental explants, the collected media samples were centrifuged at 8,000 xg to remove any cells or cellular debris; and then with 300 μL aliquots of the supernatant, cfDNA was extracted using a genomic DNA extraction technique (High Pure PCR Template Prep kits (Roche Applied Science)). The concentration of the isolated DNA was determined using a NanoDrop spectrophotometer (Thermo Scientific); and this concentration was used to calculate the total DNA content in the 2 mL of culture media. The combined weights of the 4 placental pieces were then used to normalize the DNA content for the amount of placental tissue; i.e. nanograms (ng) of cfDNA per milligrams (mg) of placental tissue. To determine the size of the cfDNA fragments, aliquots of extracted cfDNA were run on 1.5% agarose / Tris-acetate-EDTA (TAE) gels. To visualize the DNA bands, the gels were preloaded with GelRed dye (Biotum Corp.) and transilluminated with UV light.

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Manufacturer protocol

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