DNA Extraction Method from EDTA blood using Gentra PureGene Kit (D5000)
If the sample is 3ml of EDTA blood:
- Add 3ml of EDTA blood to a 15ml conical Falcon tube containing 9ml of RBC lysis solution. Invert several times and leave at RT for 10 mins, inverting occassionally.
- Spin at 3420rpm/10mins (2000g) to pellet the white cells. Decant off supernatant into Virkon, and vigorously vortex cells.
- Add 3ml of cell lysis solution, and pipette up and down to lyse the cells. If clumping occurs, then incubate at 37C until they have gone. Sample is now stable for 18 months at RT.
- [Add 15ul RNase, invert tube 25 times and incubate at 37C for >15mins.]
- Cool sample to RT (put in fridge for 3 mins) and add 1ml of protein precipitation solution. Vortex rapidly for 20secs, then spin at 3420rpm/10mins (2000g) to pellet protein.
- Decant supernatant into a tube containing 3ml of 100% isopropanol. Invert tube 50 times to form threads of DNA.
- Spin at 3420rpm/3mins (2000g) to pellet DNA. Discard isopropanol and add 1-2ml of 70% Ethanol. You can at this point transfer the pellet to a 2ml storage vial, then spin in Microfuge for 1min at maximum.
- Aspirate off as much ethanol as possible and leave to air dry for 30 minutes. For large pellets, add 250ul DNA hydration buffer. Medium sized pellets, 150-200ul, and for small pellets, 50-150ul. Best results are obtained by rehydrating the pellets overnight at RT.
If the sample is 300-500ul of EDTA blood:
- Add 300ul blood to 900ul of RBC lysis solution in a 2ml vial. Invert to mix and leave at RT for 10mins, inverting occassionally.
- Spin at 13000-16000g for 20secs to pellet white cells. Decant supernatant into Virkon and vortex tube vigorously.
- Add 300ul cell lysis solution, pipette up and down to lyse cells. If there are clumps, put at 37C until they are gone. Sample is now stable for 18 months at RT.
- Add 1.5ul RNase, invert 25 times to mix and incubate at 37C for >15mins.
- Cool sample to RT and add 100ul protein precipitation solution. Vortex vigorously for 20secs and spin at 13000-16000g for 3mins.
- Aspirate off supernatant into a clean 2ml vial containing 300ul 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 300-1000ul 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.
- For a large pellet add 150ul DNA hydration solution, for a medium size pellet 100ul, and for a small pellet 50ul. Leave to rehydrate overnight at RT.