DNA Extraction and Sequencing

Protocol: Isolation of Genomic DNA from Tissues （QIAamp DNA Micro）
This protocol is for isolation of genomic DNA from less than 10 mg tissue.
Important points before starting
■ Perform all centrifugation steps at room temperature (15–25°C)
■ If isolating DNA from very small amounts of tissue, carrier RNA is required.
■ Prepare tissue samples on a cold surface (e.g., a glass, steel, or aluminum plate
placed on top of a block of dry ice).
■ If using frozen tissue, ensure that the sample does not thaw out before addition of
Buffer ATL in step 2.

Things to do before starting
■ Equilibrate Buffer AE or distilled water for elution to room temperature (15–25°C).
■ Set a thermomixer or heated orbital incubator to 56°C for use in step 4. If a
thermomixer or heated orbital incubator is not available, a heating block or water
bath can be used instead.
■ If Buffer AL or Buffer ATL contains precipitates, dissolve by heating to 70°C with
gentle agitation.
■ Ensure that Buffers AW1 and AW2 have been prepared according to the
instructions.

Procedure
1. Transfer a tissue sample of less than 10 mg in weight to a 1.5 ml microcentrifuge
tube (not provided).
2. Immediately add 180 μl Buffer ATL, and equilibrate to room temperature
(15–25°C).
3. Add 20 μl proteinase K and mix by pulse-vortexing for 15 s.
4. Place the 1.5 ml tube in a thermomixer or heated orbital incubator, and incubate
at 56°C overnight until the sample is completely lysed.
For small amounts of tissue, lysis is complete in 4–6 h, but best results are achieved
after overnight lysis.

5. Add 200 μl Buffer AL, close the lid, and mix by pulse-vortexing for 15 s.
To ensure efficient lysis, it is essential that the sample and Buffer AL are
thoroughly mixed to yield a homogenous solution.
Note: If carrier RNA is required (see page 13), add 1 μg dissolved carrier RNA
to 200 μl Buffer AL. Note that carrier RNA does not dissolve in Buffer AL. It must
first be dissolved in Buffer AE and then added to Buffer AL.
6. Add 200 μl ethanol (96–100%), close the lid, and mix thoroughly by
pulse-vortexing for 15 s. Incubate for 5 min at room temperature (15–25°C).
Note: If room temperature exceeds 25°C, cool the ethanol on ice before adding
to the tube.
7. Briefly centrifuge the 1.5 ml tube to remove drops from inside the lid.

8. Carefully transfer the entire lysate from step 7 to the QIAamp MinElute column
(in a 2 ml collection tube) without wetting the rim. Close the lid, and centrifuge at
6000 x g (8000 rpm) for 1 min. Place the QIAamp MinElute column in a clean
2 ml collection tube, and discard the collection tube containing the flow-through.
If the lysate has not completely passed through the membrane after centrifugation,
centrifuge again at a higher speed until the QIAamp MinElute column is empty.
9. Carefully open the QIAamp MinElute column and add 500 μl Buffer AW1 without
wetting the rim. Close the lid, and centrifuge at 6000 x g (8000 rpm) for 1 min.
Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the
collection tube containing the flow-through.
10. Carefully open the QIAamp MinElute column and add 500 μl Buffer AW2 without
wetting the rim. Close the lid, and centrifuge at 6000 x g (8000 rpm) for 1 min.
Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the
collection tube containing the flow-through.

11. Centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min to dry the membrane
completely.

12. Place the QIAamp MinElute column in a clean 1.5 ml microcentrifuge tube (not
provided) and discard the collection tube containing the flow-through. Carefully
open the lid of the QIAamp MinElute column and apply 20–100 μl Buffer AE or
distilled water to the center of the membrane.

13. Close the lid and incubate at room temperature (15–25°C) for 1 min. Centrifuge at
full speed (20,000 x g; 14,000 rpm) for 1 min.

Incubating the QIAamp MinElute column loaded with Buffer AE or water for 5 min
at room temperature before centrifugation generally increases DNA yield.