Lidstrom:Genomic DNA extraction

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Revision as of 13:50, 20 February 2013 by Janet B. Matsen (Talk | contribs)
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Genomic DNA extractionPROTOCOL DNA extraction Please wear gloves at all times

  1. Add 180 µl Buffer ALT to a clean 1.5ml tube;l
  2. Add 20 µl proteinase K and mix
  3. Scoop cell biomass from Petri-plate and add it to ATL/ proteinase K mixture. Mix well;
  4. Incubate at 56oC for 3 h or overnight
  5. Add 200 µl Buffer AL to the sample, mix by pulse-vortexing for 15 s, and incubate at 70 °C for 10 min.
  6. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from inside the lid.
  7. Add 200 µl ethanol to the sample, and mix by pulse-vortexing for 15 s.
  8. 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.
  9. 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.
  10. Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate.
  11. 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.
  12. Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate
  13. Centrifuge at full speed (14,000 rpm) for 2 min.
  14. Place the SpinColumn in a clean 1.5 ml tube and discard the collection tube
  15. 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.
  16. Your DNA sample is now in the 1.5ml tube (should be 200ul). Discard the Spin Column.
  17. Measure DNA concentration on nanoDrop
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