Preparation and processing of pulsed field gel electrophoresis

1. Collect 10ml whole blood and add 30ml cell lysate. Put it in an ice bath for at least 20 minutes until the red blood cells are completely dissolved.

Centrifuge at 2.2000 rpm for 65438 00 minutes, remove the red supernatant, wash the cells with cell lysate again, and then resuspend the cells with PBS.

3. Dilute the single cell suspension, and take a small part to count the cells with neubauer cavity.

4. Resuspension the cells with PBS to achieve the ratio of 1 10,000 cells in 40μl PBS (1 10,000 diploid mammals contain about 10μ g genomic DNA).

5. Prepare 2% low-melting agarose solution with PBS and keep it at 50℃.

6. Mix the cell suspension with the same volume of agarose solution (65438 0 ml each) at room temperature, and immediately pour it into the gel block mold.

7. Let the agarose solidify after standing for 20 minutes, take the gel block out of the mold with a sterile plastic cup (generally used for sterilization) and put it in protease buffer, and add 2mg/ml protease K. ..

8. Preserve the proteinase K buffer with gel block at 50℃ for 2 ~ 3 days. Each Falcon tube containing 50 ml protease buffer can hold at most 100 gel blocks.

9. After being digested by protease K, the gel block can be stored in this buffer or 0.5mol/L EDTA solution at 4℃.

10. In addition, the step of washing the gel block with autoclaved TE buffer was continued several times.

1 1. Put the gel block into Falcon test tube containing TE and 0.04 mg/ml PMSF solution to inactivate the residual protease K. ..

12. Wash the gel block several times with TE solution at room temperature, and put the gel block into another clean test tube, which can be directly used for enzyme digestion reaction or 0.5 mol/L..

EDTA, (pH8.0)4℃ to store the gel block.

13. If the gel block is stored with EDTA, it should be washed with TE solution for 30 min×2 times at room temperature after being taken out.