DNA Extraction Method from Cultured cells using Gentra PureGene Kit (D5000)
If the sample is 3-5x106 cultured cells:
- Add 3-5 million cells to a 2ml vial and spin at 13000-16000g for 5secs to pellet cells. Discard supernatant into Virkon leaving 20-40ul residual liquid. Vortex tube vigorously.
- Add 600ul cell lysis solution, pipette up and down to lyse cells. Incubate at 37C if clumps are visible. Sample is now stable for 18 months at RT.
- Add 3ul RNase, invert 25 times to mix and incubate at 37C for >15mins.
- Cool sample to RT and add 200ul protein precipitation solution. Vortex vigorously for 20secs and spin at 13000-16000g for 3mins.
- Aspirate off supernatant into a clean 2ml vial containing 600ul of 100% isopropanol. Invert tube 50 times to form threads of DNA.
- Spin at 13000-16000g for 1min to pellet DNA. Discard isopropanol and add 600ul of 70% ethanol. Invert tube several times to wash the pellet then spin at 13000-16000g for 1min.
- Decant off as much ethanol as possible using micropipette tips, and air dry for 30mins.
- Add 100ul DNA hydration solution and leave to rehydrate overnight at RT.