DNA提取 z-bo 2020-10-14 307

　　(1) Remove the material, grind it into powder with liquid nitrogen, and then quickly transfer it to a 1.5 ml Eppendorff tube;

　　(2) Add 800μl CTAB extraction buffer and mix thoroughly (CTAB is preheated in a 65°C water bath) shake gently every 5 minutes, and then centrifuge at 12000r/min for 15 minutes after 20 minutes;

　　(3) Carefully aspirate the supernatant and the same amount of phenol:chloroform (400μl each time). ) Add the solution, mix well, and centrifuge at 12000r/min for 10 minutes at 4°C.

　　(4) Carefully aspirate the supernatant, add the same amount of chloroform, mix well and centrifuge at 12000r/min for 10 minutes at 4°C;

　　(5) Repeat (4) once or twice to prevent the protein layer from appearing.

　　(6) Take the supernatant and let it stand at -20°C for 1 hour, 4°C, 12000r/min and centrifuge for 10 minutes;

　　(7) Discard the supernatant and 70% of the precipitate and wash twice with ethanol;

　　(8) After drying at room temperature (usually 5-15 minutes), dissolve in 30-50μl DEPC deionized water and -20°C or -70 for later use and store at °C.

　　2.2.1 Sample processing:

　　(1) Cell culture: Collect 1-5 x 107 cells, transfer them to a 1.5 ml centrifuge tube, add 1 ml of Trisol, mix well, and then leave it at room temperature for 5 minutes.

　　(2) Tissue: Take 50-100 mg of tissue (fresh or stored in -70°C and liquid nitrogen), place it in a 1.5 ml centrifuge tube, add 1 ml of triol for homogenization, and wait 5 times at room temperature

　　2.2.2 Extraction steps:

　　(1) Add 0.2 ml of chloroform, shake for 15 seconds, and leave for 2 minutes.

　　(2) Centrifuge at 4°C, 12000g×15 minutes to remove the supernatant.

　　(3) Add 0.5 ml of isopropanol, gently mix the liquid in a test tube, and leave it at room temperature for 10 minutes.

　　(4) Centrifuge at 12000g×10 minutes at 4°C and discard the supernatant.

　　(5) Add 1 ml of 75% ethanol, and then gently wash the precipitate. Discard the supernatant at 4°C, 7500g x 5 minutes. (6) Dry and add appropriate amount of DEPCH2O to dissolve it (10-15 minutes at 65°C).