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SQ Blood Mini Protocol for 50ul Clotted Blood

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617

实验试剂

 

Regents to be supplied by user

1. Proteinase K (20mg/ml)

2. Isopropanol

3. 70% ethanol

实验设备

 

Equipments to be supplied by user

1. Microcentrifuge capable of 14,000 x g

2. Nuclease-free 1.5 ml microcentrifuge tubes

3. Water Bath preset at 37°C

实验步骤

 

1. Transfer the 50ul blood include any liquid residual into a 1.5 ml centrifuge tube.

2. Add 550ul WTL Buffer and pipet up an down a few times to mix.

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

4. Incubate at 55°C for 1 hour to overnight until clots has dissolved.

5. Place the tube on ice for 1 minute.

6. Add 3 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 minute. Add 200 ul PCP buffer to the cell lysate. Vortex vigorously at high speed for 30 seconds to mix. Some protein clumps may be visible after vortexing. Incubate in ice for 5 minutes.

8. 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 this step.

9. Transfer the supernatant to a new nuclease-free 2.0 ml centrifuge tube containing 600ul of 100% isopropanol. If the DNA yield is expected to be lower than 2ug, add 2ul of glycogen (20mg/ml) per sample.

10. Gently mix the solution by inverting the tube 30-40 times. Centrifuge at 14,000 x g for 1 minute at room temperature. DNA will be visible as a small white pellet.

11. Pour of the supernatant and drain the tube briefly on a clean absorbent paper towel. Add 600ul 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 20 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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