DNA extraction method (separation of DNA from whole blood)

Isolate DNA from whole blood

1. The required solution:

1

PBS buffer

NaCl

8 g

KCl

0.2 g

Na2HPO4

1.44 g

KH2PO4

0.24 g

Add ultrapure water to a volume of 1000 mL, adjust the pH to 7.4, autoclave, and store at 4 ℃.

2

DNA extract

Tris (pH8.0) / MW 121.14

0.6057 g

10 mmol / L

EDTA / MW 372.24

18.612 g

0.1 mol / L

SDS / MW 288.38

2.5 g

0.5%

Add ultrapure water to 500 mL, adjust pH to 8.0, autoclave, and store at 4 ℃.

Put it in a 37 ° C water bath before use to dissolve it completely.

3

Proteinase K

20 mg / mL

Store at 20 ℃

4

Tris saturated phenol

5

Chloroform

6

Absolute ethanol

Store at 20 ℃ before use

7

70% ethanol

8

TE buffer

Tris / MW 121.14

0.6057 g

10 mmol / L

EDTA / MW 372.24

0.1861 g

1 mmol / L

Add ultrapure water to 500 mL, adjust pH to 8.0, autoclave, and store at 4 ℃.

Second, the main equipment required

Ice maker, refrigerator, freezing low temperature ultracentrifuge, pipette, constant temperature water bath

3. Operation steps

1. Frozen blood samples are melted in a room temperature water bath;

2. Transfer 1 mL of whole blood into a sterile 2 mL centrifuge tube;

3. Add an equal volume (1 mL) of PBS buffer and gently shake for 15 min;

4. Centrifuge at 3500 g for 10 min at room temperature;

5. Discard the supernatant with a pipette and repeat steps 3 and 4 until the supernatant is transparent and the precipitate is colorless;

6. Add 1 mL of DNA extraction solution to the centrifuge tube and gently shake to suspend the cell pellet;

7. 37 ℃ water bath for 1 h;

8. Add 3 μL of proteinase K (final concentration is 60 μg / mL) and mix well;

9. Incubate at 55 ℃ overnight (about 16 h) in a constant temperature water bath until the cell pellet is completely digested and the solution is clear;

10. Cool the reaction solution to room temperature, add 1 volume (1 mL) of Tris saturated phenol, and place it on ice and shake gently for 20 min;

11. Centrifuge at 12,000 g for 10 min at 4 ℃;

12. Use a pipette to transfer the upper aqueous phase to another sterilization centrifuge tube;

13. Add 0.5 volumes (0.5 mL) of phenol and 0.5 volumes (0.5 mL) of chloroform, place on ice and shake gently for 20 min;

14. Centrifuge at 12,000 g for 10 min at 4 ℃;

15. Transfer the upper aqueous phase to another sterilized centrifuge tube with a pipette;

16. Add 1 volume (1 mL) of chloroform, place on ice and shake gently for 20 min;

17. Centrifuge at 12,000 g for 10 min at 4 ℃;

18. Pipette the upper aqueous phase into another sterilization centrifuge tube;

19. Add twice the volume of pre-cooled absolute ethanol (? 20 ℃), gently shake the bottom of the mouth many times until the DNA precipitates, and then stand at -20 ℃ for 30 min;

20. Use a glass hook to hook the DNA pellet into a new sterilized centrifuge tube, or centrifuge at 12,000 g for 10 min at 4 ℃, and discard the ethanol;

21. Add 1 mL of 70% ethanol and shake gently for 10 min;

22. Centrifuge at 12,000 g for 10 min at 4 ℃, discard ethanol and repeat rinsing once;

23. Vacuum drying or evaporating ethanol at room temperature;

24. According to the amount of DNA, add 100-300 μL of ultrapure water and store at 4 ℃ until the DNA is completely dissolved. After measuring the concentration with a spectrophotometer, store at -80 ℃.

DNA isolation from tissue samples

1. The required solution:

1

SE buffer

NaCl / 58.44

0.7305 g

25 mmol / L

EDTA / MW 372.24

13.959 g

75 mmol / L

SDS / MW 288.38

5 g

1%

Add ultrapure water to 500 mL, adjust pH to 8.0, autoclave, and store at 4 ℃.

2

Proteinase K

20 mg / mL

Store at 20 ℃

3

6 mol / L NaCl

Preheat at 65 ℃ before use

4

Chloroform

5

Absolute ethanol

Store at 20 ℃ before use

6

70% ethanol

7

TE buffer

Tris / MW 121.14

0.6057 g

10 mmol / L

EDTA / MW 372.24

0.1861 g

1 mmol / L

Add ultrapure water to 500 mL, adjust pH to 8.0, autoclave, and store at 4 ℃.

Second, the main equipment required

Ice maker, refrigerator, freezing low temperature ultracentrifuge, pipette, constant temperature water bath

3. Operation steps

1. Weigh 10 mg of tissue sample into a 1.5 mL centrifuge tube;

2. Use a suitable clean glass rod or plastic rod to crush the tissue along the centrifuge tube wall;

3. Add 500 μL SE buffer;

4. Add 5 μL of proteinase K (20 mg / mL) to each sample;

5. The sample is placed in a water bath thermostat at 65 ℃ for 12-16 h;

6. Take out the sample, add 180 μL of pre-heated 6 mol / L NaCl at 65 ℃, mix thoroughly (shaking 15 times at the bottom of the mouth);

7. Place the sample on ice, add 700 μL of chloroform, and shake gently for 20 min;

8. Centrifuge at 12,000 g for 10 min at 4 ℃;

9. Transfer the upper aqueous phase to another sterilized centrifuge tube with a pipette;

10. Repeat steps 7, 8, and 9 once;

11. Add 2 volumes of pre-cooled absolute ethanol (? 20 ℃), shake gently at the bottom of the mouth many times until the DNA precipitates, and then leave at 20 ℃ for 30 min;

12. Use a glass hook to hook the DNA pellet into a new sterilized centrifuge tube, or centrifuge at 12,000 g for 10 min at 4 ℃, and discard the ethanol;

13. Add 1 mL of 70% ethanol and shake gently for 10 min;

14. Centrifuge at 12,000 g for 10 min at 4 ℃, discard ethanol and repeat rinsing once;

15. Vacuum dry or make ethanol evaporate clean at room temperature;

16. According to the amount of DNA, add 100-300 μL of ultrapure water and store at 4 ℃ until the DNA is completely dissolved. After measuring the concentration by spectrophotometer, store at -80 ℃.

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