实验概要

The E.Z.N.A.^®  SQ Blood DNA Kit is designed for isolating high molecular weight  genomic DNA from fresh, frozen or anticoagulated whole blood. The method  can also be used for preparation of genomic DNA from buffy coat, bone  marrow or cultured cells. The kit allows single or multiple,  simultaneous processing of samples in under 90 minutes. There is no need  for phenol/chloroform extractions, and time-consuming steps such as  CsCl gradient ultracentrifugation are eliminated. DNA purified using the  E.Z.N.A.^® SQ Blood DNA method is ready for applications such as PCR*, Southern blotting, and restriction digestion.

The E.Z.N.A.^® SQ Blood DNA Kit uses a highly efficient  solution based system to provide a convenient, fast, reliable and  non-toxic method to isolate high molecular weight genomic DNA from whole  blood or buffy coat. Red blood cells are first lysed with ERL buffer,  followed by lysis of the white blood cells and their nuclei in the WTL  Buffer. Cellular proteins are removed by precipitation and high  molecular weight genomic DNA will remain in solution. High quality  genomic DNA is then purified by isopropanol precipitation.

主要试剂

1. Isopropanol (100%)

2. 70% ethanol

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

主要设备

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 15 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 ddH₂O 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 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.

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. Add1/ 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 5 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 the 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.