DNA Extraction Method from Cultured cells using Gentra PureGene Kit (D5000)

If the sample is 3-5x106 cultured cells:

  1. 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.
  2. 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.
  3. Add 3ul RNase, invert 25 times to mix and incubate at 37C for >15mins.
  4. Cool sample to RT and add 200ul protein precipitation solution. Vortex vigorously for 20secs and spin at 13000-16000g for 3mins.
  5. Aspirate off supernatant into a clean 2ml vial containing 600ul of 100% isopropanol. Invert tube 50 times to form threads of DNA.
  6. 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.
  7. Decant off as much ethanol as possible using micropipette tips, and air dry for 30mins.
  8. Add 100ul DNA hydration solution and leave to rehydrate overnight at RT.