SQBloodMiniProtocolfor50ulClottedBlood

实验概要

This protocol is designed for isolating genomic DNA from 0.5-1 million cultured cells. For larger or smaller amounts of starting cell numbers, please use protocol in the E.Z.N.A^® SQ Tissue DNA Kit.

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 2.0 ml microcentrifuge tubes

3. Water Bath preset at 37°C

实验步骤

1. Harvest the cells and transfer them with salt balanced buffer (such as PBS) to a 2.0 ml microcentrifuge tube. For adherent cells, trypsinize the cells before harvesting.

2. Centrifuge at 14,000 x g for 10 seconds to pellet the cells. Remove the cells and leave behind about 10ul residue liquid.

3. Vortex the cells to resuspend the cells in the residue liquid. Make no cell clumps visible at this point.

4. Add 150ul of WTL Buffer to the resuspended cells and mix by pipetting. 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 1 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.

6. Cool the sample to room temperature. Add 50ul 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 on ice for 5 minutes.

7. Centrifuge at max speed for 3 minutes at room temperature. The precipitated protein will form a tight pellet. If the pellet is not tight or visible, incubate the tube in ice for 5 minutes and repeat Step 8.

8. Transfer the supernatant to a new nuclease-free 2.0 ml centrifuge tube containing 150ul of 100% isopropanol. Gently mix the solution by inverting the tube 30-40 times.

9. Centrifuge at 14,000 x g for 1 minute at room temperature. DNA will be visible as a small white pellet.

10. Pour out the supernatant and drain the tube briefly on a clean absorbent paper towel. Add 150ul of 70% ethanol and invert the tube a few times to wash the DNA pellet.

11. 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.

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

13. Add 50 ul of DNA rehydration solution (Buffer EB) and vortex for 1 minutes to mix.

14. Incubate sample at 65°C for 10 min. Some samples may need to incubate at 65°C for 1 hour to rehydrate DNA. Store DNA at 2-8°C. For long-term storage, store at -20°C.