Isolating Genomic DNA from Whole Blood

This procedure can be performed for samples of 300µl or 3ml of blood. When steps differ in the volumes, both versions will be presented in the style below. Start with the following step.

• 300µl sample: Add 900µl of Cell Lysis Solution to a sterile 1.5ml microcentrifuge tube.
• 3ml sample: Add 9.0ml of Cell Lysis Solution to a sterile 15ml centrifuge tube.

Blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Gently rock the tube of blood until thoroughly mixed, then transfer blood to the tube containing the Cell Lysis Solution. Invert the tube 5–6 times to mix.

Incubate the mixture for 10 minutes at room temperature (invert 2–3 times once during the incubation) to lyse the red blood cells.

• 300µl sample: Centrifuge at 13,000–16,000 × g for 20 seconds at room temperature.
• 3ml sample: Centrifuge at 2,000 × g for 10 minutes at room temperature.

Remove and discard as much supernatant as possible without disturbing the visible white pellet.

• 300µl sample: Approximately 10–20µl of residual liquid will remain in the 1.5ml tube.
• 3ml sample: Approximately 50–100µl of residual liquid will remain in the 15ml tube.

If blood sample has been frozen, repeat Steps 1–4 until pellet is white. There may be some loss of DNA from frozen samples.

Note: Some red blood cells or cell debris may be visible along with the white blood cells. If the pellet appears to contain only red blood cells, add an additional aliquot of Cell Lysis Solution after removing the supernatant above the cell pellet, and then repeat Steps 3–4.

Vortex the tube vigorously until the white blood cells are resuspended (10–15 seconds). Completely resuspend the white blood cells to obtain efficient cell lysis.

Add Nuclei Lysis Solution to the tube containing the resuspended cells. Pipet the solution 5–6 times to lyse the white blood cells.

• 300µl sample: 300µl Nuclei Lysis Solution.
• 3ml sample: 3.0ml Nuclei Lysis Solution.

The solution should become very viscous. If clumps of cells are visible after mixing, incubate the solution at 37°C until the clumps are disrupted. If the clumps are still visible after 1 hour, add additional Nuclei Lysis Solution and repeat the incubation.

• 300µl sample: 100µl Nuclei Lysis Solution.
• 3ml sample: 1.0ml Nuclei Lysis Solution.

Add RNase Solution to the nuclear lysate, and mix the sample by inverting the tube 2–5 times.

• 300µl sample: 1.5µl RNase Solution.
• 3ml sample: 15µl RNase Solution.

Incubate the mixture at 37°C for 15 minutes, then cool to room temperature.

Add Protein Precipitation Solution to the nuclear lysate.

• 300µl sample: 100µl Protein Precipitation Solution.
• 3ml sample: 1.0ml Protein Precipitation Solution.

Vortex vigorously for 10–20 seconds. Small protein clumps may be visible after vortexing.

Note: If additional Nuclei Lysis Solution was added in Step 6, add a total of 130µl Protein Precipitation Solution for 300µl sample volume and 1.3ml Protein Precipitation Solution for 3ml sample volume.

• 300µl sample: Centrifuge at 13,000–16,000 × g for 3 minutes at room temperature.
• 3ml sample: Centrifuge at 2,000 × g for 10 minutes at room temperature.

A dark brown protein pellet should be visible.

Transfer the supernatant to a clean microcentrifuge tube containing room-temperature isopropanol.

• 300µl sample: 1.5ml tube with 300µl isopropanol.
• 3ml sample: 15ml tube with 3ml isopropanol.

Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.

Gently mix the solution by inversion until the white thread-like strands of DNA form a visible mass.

• 300µl sample: Centrifuge at 13,000–16,000 × g for 1 minute at room temperature.
• 3ml sample: Centrifuge at 2,000 × g for 1 minute at room temperature.

The DNA will be visible as a small white pellet.

Decant the supernatant, and add one sample volume of room-temperature 70% ethanol to the DNA. Gently invert the tube several times to wash the DNA pellet and the sides of the microcentrifuge tube. Centrifuge as in the previous step.

Carefully aspirate the ethanol using either a drawn Pasteur pipette or a sequencing pipette tip. The DNA pellet is very loose at this point, and care must be used to avoid aspirating the pellet into the pipette. Invert the tube on clean absorbent paper, and air-dry the pellet for 10–15 minutes.

• 300µl sample: Add 100µl DNA Rehydration Solution to the tube.
• 3ml sample: Add 250µl DNA Rehydration Solution to the tube.

Rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature or 4°C.

Store the DNA at 2–8°C.