Lidstrom:Genomic DNA extraction
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Genomic DNA minikit extraction PROTOCOL
- From Marina K.
- Please wear gloves at all times
- Add 180 µl Buffer ALT to a clean 1.5ml tube
- Add 20 µl proteinase K and mix
- Scoop cell biomass from Petri-plate and add it to ATL/ proteinase K mixture. Mix well;
- Incubate at 56oC for 3 h or overnight
- Add 200 µl Buffer AL to the sample, mix by pulse-vortexing for 15 s, and incubate at 70 °C for 10 min.
- Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from inside the lid.
- Add 200 µl ethanol to the sample, and mix by pulse-vortexing for 15 s.
- Carefully apply the mixture from step 4 (including the precipitate) to the Spin Column (in a 2 ml collection tube). Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the Spin Column in a clean 2 ml collection tube (provided),and discard the tube containing the filtrate.
- Carefully open the Spin Column and add 500 µl Buffer AW1. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min.
- Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate.
- Carefully open the Spin Column and add 500 µl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (14,000 rpm) for 3 min.
- Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate
- Centrifuge at full speed (14,000 rpm) for 2 min.
- Place the SpinColumn in a clean 1.5 ml tube and discard the collection tube
- Carefully open the Spin Column and add 100 µl Buffer AE. Incubate at room temperature for 2 min, and then centrifuge at 12 000 rpm for 1 min. Repeat this step.
- Your DNA sample is now in the 1.5ml tube (should be 200ul). Discard the Spin Column.
- Measure DNA concentration on nanoDrop
Comments
- Use approximately "a booger" worth of cells
- If your cells are on a plate, wash them off with their growth medium and pellet in a centrifgue.
- Aaron Puri incubated cells + ALT + proteinase K for 6 hours and got 40 ng/uL.