DNA ExtractionDNA Extraction
Procedure Page
Qiagen DNeasy Protocol for Animal Tissues - simplified from the directions included in the kit.
In this protocol, the animal's parts are ground up manually and digested with enzymes. The "parts" must then be separated from the DNA. This is made easier with Qiagen's system of washes and mini column filter.
μl of Buffer ATL and homogenize thoroughly.1. Cut up to 25 mg of tissue into small pieces and place in a 1.5 mL tube.
2. Add 90
3. Add another 90 μl of Buffer ATL; homogenize and/or vortex.
4. Add 20 μl of Proteinase K and vortex.
5. Incubate at 55 C in a water bath for at least 1 hr.
6. Vortex for 15 seconds.
7. Add 200 μl Buffer AL; vortex.
8. Incubate at 70 C in a heat block for 10 minutes.
9. Add 200 μl ethanol (96 - 100%); vortex.
10. First wash/filter:
Pipet all of the mixture into a DNeasy mini column which contains a filter. The mini column should be sitting in a 2 mL collection tube.11. Centrifuge at 8000rpm for 1 min. Save the mini column but discard the collection tube and the liquid collected in it.
12. Place the mini column in a new collection tube.
13. Second wash/filter: Add 500
μl Buffer AW1 to the mini column.14. Centrifuge at full speed for 3 minutes. Save the mini column but discard the collection tube and the liquid collected in it.
15. Place the mini column in 1.5 mL tube which will act as a collection tube.
16. First DNA Collection
: Add 200 μl of Buffer AE to the mini column.17. Centrifuge at 8000rpm for 1 min. The DNA will be pulled through the filter into the collection tube. Save and set aside the 1.5 mL collection tube and the liquid in it
18. Place the mini column in another 1.5 mL tube.
19. Second DNA Collection
: Add 200 μl of Buffer AE to the mini column.20. Centrifuge at 8000rpm for 1 min. The last bits of DNA will be pulled through the filter into the collection tube.
21. Combine the contents of the two 1.5 mL collection tubes. This is your concentrated DNA, and should be stored as backup stock.
22. Create a usable, more dilute stock of DNA: Combine 20 μl of concentrated DNA and 180 μl distilled water in a separate 1.5 mL or 0.2 mL tube.