Total DNA is required for real-time PCR amplification to determine mtDNA
copy number and to analyze variants by allele-specific PCR.
Total DNA is extracted using the Puregene DNA Isolation Kit, (Flowgen, UK).
1. Pellet hESCs at 13,000g for 5 s.
2. Remove the supernatant and resuspend the hESCs in 10-20 |L of residual supernatant and vortex vigorously to ensure thorough resuspension.
3. Extract the DNA by resuspending the cells in 300 ||L of cell lysis solution.
4. Add 1.5 |L of RNase A solution, invert multiple times and incubate at 37°C for 5-20 min.
5. Precipitate the proteins by cooling the sample on ice for 1 min. Add 100 ||L of protein precipitation solution and vortex at high speed for 20 s. Centrifuge the precipitated proteins at 13,000g for 1 min.
6. Precipitate the DNA by pouring the supernatant containing the DNA into a sterile 1.5-mL microcentrifuge tube already containing 300 |L 100% isopropanol and mix by repeated inversion. Centrifuge the mixture at 13,000g for 1 min to form a small, white pellet.
7. Pour off the supernatant and add 300 |L 70% ethanol. Repeat the centrifugation at 13,000g for 1 min. The ethanol is then poured off and the tube inverted and placed on absorbent paper for 10-15 min before rehydration.
8. Recover the DNA in 50 |L of autoclaved sterile ddH2O.
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