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    SQ Blood DNA Maxi Protocol for 4-10 ml whole blood

    互联网

    1142

    实验试剂

     

    1. Isopropanol (100%)

    2. 70% ethanol

    3. Using different volume of Solution according blood volume as following:

    实验设备

     

    1. Centrifuge capable of 2,000 x g

    2. Nuclease-free 15 ml microcentrifuge tubes

    3. Water baths preset at 37°C

    4. Paper towels

    实验步骤

     

    1. Add one volume of whole blood (or bone marrow) to a nuclease-free 50 ml centrifuge tube containing 3 volume of Buffer ERL. Mix by inverting the tube a few times. Incubate 5 minutes at room temperature. Invert the tube several times during incubation.

    NOTE: ERL Buffer is supplied as a 10x concentrate and must be diluted with ddH2 O according to bottle label.

    2. Centrifuge at 2,000 x g for 5 minutes at room temperature. Remove and discard as much supernatant as possible without disturbing the visible white pellet. Leave about 0.1 volume of residue liquid in the tube. If the blood sample has been frozen, repeat Steps 1-2 until the pellet is white.

    Note: If some red blood cells or cell debris are still visible along with the white blood cell pellet, resuspend the white blood cell pellet and mix with 2 volumes ERL Buffer. Incubate 2 min at room temperature. Then pellet the white blood cells by repeating Step 2.

    3. Vortex the tube vigorously until the white blood cells are completely resuspended.

    4. Add one volume of Buffer WTL to the tube containing the resuspended cells. Pipet up and down to lyse the cells. The solution should become very viscous. If cell clumps are visible after mixing, incubate the solution at 37oC until the clumps cannot be seen.

    5. (Optional) Add correct volume of RNase A solution to the cell lysate. Mix the sample by inverting the tube 20-25 times. Incubate the mixture at 37°C for approximately 10 minutes.

    6. Cool the sample to room temperature. Add 1/3 Volume of Buffer PCP to the cell lysate. Vortex vigorously at high speed for 30 seconds to mix. Some protein clumps may be visible after vortexing.

    7. Centrifuge at 2,000 x g or higher for 5 minutes at room temperature. The precipitated protein will form a tight, dark brown pellet. If the pellet is not tight or visible, incubate the tube on ice for 5 minutes, and then repeat Step 7.

    8. Transfer the supernatant to a new nuclease-free 15 ml centrifuge tube containing one volume of 100% isopropanol. Do not transfer the protein pellet.

    9. Gently mix the solution by inverting the tube 40-50 times.

    10. Centrifuge at 2,000 x g for 3 minutes at room temperature. DNA will be visible as a small white pellet.

    11. Pour out the supernatant and drain the tube briefly on an absorbent paper towel. Add one volume of 70% ethanol and invert the tube a few times to wash the DNA pellet.

    12. Centrifuge at 2,000 x g for 2 minutes at room temperature. Carefully pour out the ethanol. Pellet may be very loose at this point, so pour slowly and be careful not to pour out the pellet.

    13. Invert the tube on a clean adsorbent paper towel and air dry the pellet for 10-15 minutes.

    14. Add 0.1 volume of Buffer EB and vortex for 1 minute to mix.

    15. Incubate sample at 65°C for 1 hour to rehydrate DNA. Gently shake tube several times during incubation to disperse DNA. Some samples may need to incubate at 65°C overnight to rehydrate DNA. Store DNA at 2-8°C or -20°C.

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