实验概要
The buffy coat fraction of whole blood is enriched with WBC, and usually gives at least 5-fold more DNA than the same volume of blood. To prepare buffy coat from fresh whole blood, simply centrifuge the sample at 3,000-4,000 x g for 10 min at room temperature. Three layers should be obtained, with plasma in the upper layer, leucocytes in the middle layer (buffy coat), and erythrocytes in bottom layer. Carefully aspirate the plasma, making sure not to disturb the layer of concentrated leukocytes. The buffy coat can be drawn off with a pipette and used directly in the E.Z.N.A.® SQ Blood DNA Protocol, or frozen at -70°C for storage.
主要试剂
Regents to be supplied by user
1. Isopropanol
2. 70% ethanol
主要设备
Equipments to be supplied by user
1. Microcentrifuge capable of 14,000 x g
2. Nuclease-free 5.0 ml microcentrifuge tubes
3. Water Bath preset at 37°C
实验步骤
1. Add 75-120 μl buffy coat preparation (prepared from 1.5 ml whole blood) to a nuclease-free 2 ml microcentrifuge tube containing 400ul ERL Buffer. Mix by inverting the tube a few times. Incubate 5 minutes at room temperature. Invert the tube several times during incubation.
2. Centrifuge at 14,000 x g for 30 seconds at room temperature. Remove and discard as much supernatant as possible without disturbing the visible white pellet. Leave about 15ul 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. Add 1.3 ml WTL Buffer 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 37°C until the clumps cannot be seen.
4. (Optional) Add 5 ul RNase A solution to the cell lysate. Mix the sample by inverting the tube 20-25 times. Incubate the mixture at 37°C for 5-10 minutes.
5. Cool the sample to room temperature. Add 433ul PCP Buffer to the cell lysate.
6. Vortex vigorously at high speed for 30 seconds to mix. Some protein clumps may be visible after vortexing. Incubate in ice for 5 minutes.
7. Centrifuge at max speed for 3 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 in ice for 5 minutes and repeat Step 7.
8. Transfer the supernatant to a new nuclease-free 2.0 ml centrifuge tube containing 1.3 ml of 100% isopropanol.
9. Gently mix the solution by inverting the tube 30-40 times.
10. Centrifuge at 14,000 x g for 1 minute at room temperature. DNA will be visible as a small white pellet.
11. Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel. Add 1.3 ml of 70% ethanol and invert the tube a few times to wash the DNA pellet.
12. Centrifuge at 14,000 x g for 2 minutes at room temperature. Carefully pour off the ethanol. Pellet may be very loose at this point, so pour slowly and watch the pellet.
13. Invert the tube on a clean adsorbent paper towel and air dry the pellet for 10-15 minutes.
14. Add 500 ul of DNA rehydration solution (Buffer EB) and vortex for 1 minute to mix.
15. Incubate sample at 65°C for 10 min. Some samples may need to incubate at 65°C for 1 hour to rehydrate DNA.
16. Store DNA at 2-8°C. For long-term storage, store at -20°C.
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