REPLI-g UltraFast Mini Kit (100)

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Publication protocol

On-chip whole genome amplification: Whole genome amplification (WGA) of the single cell genomic DNA tethered within the micropillar array region of the microfluidic device was carried out using reagents from the REPLI-g UltraFast Mini Kit (Qiagen; Hilden, Germany). Prior to starting the reaction, 280μl of buffer D1 was made by adding 35μl of buffer DLB to 245μl of ultrapure H2O. 400μl of buffer N1 was then prepared by adding 40μl of stop solution to 360μl of ultrapure H2O. Finally, 288μl of master mix was made by adding 18μl of polymerase to 270μl Repli-G UltraFast reaction buffer. To denature the double stranded gDNA tethered on the micropillar array, buffer D1 was flowed through the device continuously at room temperature for 8 minutes. Buffer D1 was then removed and the device was flushed with buffer N1 for 15 minutes. Afterwards, both the infusion apparatus and the ten output reservoirs were emptied and washed with 100% ethanol and then ultrapure water. The infusion apparatus was then loaded with the master mix solution and pressure was set to 0.5 psi. Pressure was then held constant throughout the entire duration of the 3.5 hour reaction amplification reaction while the device was placed atop a hot-plate set to 33C. After the reaction was completed, 5μl of ultrapure water was added to the amplified DNA product at each output reservoir. The amplified genomic DNA in the reservoir was then pipette collected from each output reservoir off-chip into a polymerase chain reaction (PCR) tube. All samples were heat inactivated at 65C for 10 minutes and placed in a -20C freezer until further use. Sample yield was measured using Qubit 2.0 Fluorometer (ThermoFisher; Waltham, MA) with dsDNA HS dye kit at a 1:200 sample dilution.

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