实验概要

This protocol is designed for isolating genomic DNA from 1ml Clotted Blood.

主要试剂

Regents to be supplied by user

1. Proteinase K (20mg/ml)

2. Isopropanol

3. 70% ethanol

主要设备

Equipments to be supplied by user

1. Microcentrifuge capable of 2,000 x g

2. Nuclease-free 50 ml microcentrifuge tubes

3. Water Bath preset at 37°C

实验步骤

1. Transfer 1 ml clotted blood including any liquid residual into a 50 ml centrifuge tube.

2. Add 11 ml WTL Buffer and pipet up an down a few times to mix.

3. Add 50 ul Proteinase K solution (20mg/ml) and mix by inverting 20 times.

4. Incubate at 55°C for 3 hour to overnight until clots has dissolved.

5. Place the tube on ice for 1-2 minute.

6. Add 50 ul RNase A to the cell lysate and invert 10 time to mix throughly. Incubate the tube at 37°C for 5 minutes.

7. Place the tube on ice for 1-2 minute.

8. Add 4 ml PCP buffer to the cell lysate.

9. Vortex vigorously at high speed for 30 seconds to mix. Some protein clumps may be visible after vortexing. Incubate in ice for 10 minutes.

10. Centrifuge at 2000 x g for 10 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 this step.

11. Transfer the supernatant to a new 50 ml centrifuge tube containing 12 ml of 100% isopropanol. Add 20ul of glycogen (20mg/ml) per sample.

12. Gently mix the solution by inverting the tube 30-40 times. Centrifuge at 2000 x g for 5 minute at room temperature. DNA will be visible as a small white pellet.

13. Pour of the supernatant and drain the tube briefly on a clean absorbent paper towel. Add 12ml of 70% ethanol and invert the tube a few times to wash the DNA pellet.

14. Centrifuge at 2000 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.

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

16. Add 400 ul of DNA rehydration solution (Buffer EB) and vortex for 1 minute to mix.

17. Incubate sample at 65°C for 1 hour. Some samples may need to incubate at room temperature for overnight to rehydrate DNA. Store DNA at 2-8°C. For long-term storage, store at -20°C.