Preparation of human peripheral blood leukocyte DNA

2022-11-16 07:06:07

【Fundamental】

Preparation of leukocyte DNA from whole blood, water can directly rupture the cell membrane and nuclear membrane of red blood cells and white blood cells in the blood, release hemoglobin and nucleus; then cleave the nucleus and dissolve DNA with KI; finally remove protein with phenol/chloroform, add absolute ethanol The DNA was precipitated, and the DNA content was detected by A260/A280, and then identified by agarose gel electrophoresis.

【equipment】

Centrifuge

2. Ultraviolet spectrophotometer

【Reagents】

1.5mmol/L KI: 8.3g KI plus H2O to 10ml

2.0.9% NaCl

3. Chloroform / isoamyl alcohol (24:1)

【Steps】

1. Take 200 μl of anticoagulant and add 1 ml of water.

Centrifuge at 2.8000 rpm for 5 min and discard the supernatant.

3. 40 μl of 5 mol / L KI was added to the pellet and vortexed at 30 Sec.

4. Add 200 μl of 0.9% NaCl, then add 300 μl of chloroform/isoamyl alcohol and shake 30 Sec.

Centrifuge at 5.10 000 rpm for 5 min.

6. Carefully pipet the supernatant and add 1 ml of cold absolute ethanol.

Centrifuge at 7.10 000 rpm for 5 min and discard absolute ethanol.

8. The DNA sample is naturally dry.

9. Add the appropriate amount of H2O or TE to dissolve the DNA.

10. Nucleic acid quantitative determination and electrophoresis identification.

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